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Sample records for inhibits myosin motor

  1. Mechanism and Specificity of Pentachloropseudilin-mediated Inhibition of Myosin Motor Activity*

    PubMed Central

    Chinthalapudi, Krishna; Taft, Manuel H.; Martin, René; Heissler, Sarah M.; Preller, Matthias; Hartmann, Falk K.; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O.; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M.; Manstein, Dietmar J.

    2011-01-01

    Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC50 values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway. PMID:21680745

  2. Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

    PubMed

    Chinthalapudi, Krishna; Taft, Manuel H; Martin, René; Heissler, Sarah M; Preller, Matthias; Hartmann, Falk K; Brandstaetter, Hemma; Kendrick-Jones, John; Tsiavaliaris, Georgios; Gutzeit, Herwig O; Fedorov, Roman; Buss, Folma; Knölker, Hans-Joachim; Coluccio, Lynne M; Manstein, Dietmar J

    2011-08-26

    Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.

  3. Myosin V motor proteins

    PubMed Central

    Vale, Ronald D.

    2003-01-01

    Mammalian myosin V motors transport cargo processively along actin filaments. Recent biophysical and structural studies have led to a detailed understanding of the mechanism of myosin V, making it perhaps the best understood cytoskeletal motor. In addition to describing the mechanism, this review will illustrate how “dynamic” single molecule measurements can synergize with “static” protein structural studies to produce amazingly clear information on the workings of a nanometer-scale machine. PMID:14610051

  4. Myosin VI: a multifunctional motor.

    PubMed

    Lister, I; Roberts, R; Schmitz, S; Walker, M; Trinick, J; Veigel, C; Buss, F; Kendrick-Jones, J

    2004-11-01

    Myosin VI moves towards the minus end of actin filaments unlike all the other myosins so far studied, suggesting that it has unique properties and functions. Myosin VI is present in clathrin-coated pits and vesicles, in membrane ruffles and in the Golgi complex, indicating that it has a wide variety of functions in the cell. To investigate the cellular roles of myosin VI, we have identified a variety of myosin VI-binding partners and characterized their interactions. As an alternative approach, we have studied the in vitro properties of intact myosin VI. Previous studies assumed that myosin VI existed as a dimer but our biochemical characterization and electron microscopy studies reveal that myosin VI is a monomer. Using an optical tweezers force transducer, we showed that monomeric myosin VI is a non-processive motor with a large working stroke of 18 nm. Potential roles for myosin VI in cells are discussed. PMID:15493988

  5. A small-molecule inhibitor of T. gondii motility induces the posttranslational modification of myosin light chain-1 and inhibits myosin motor activity.

    PubMed

    Heaslip, Aoife T; Leung, Jacqueline M; Carey, Kimberly L; Catti, Federica; Warshaw, David M; Westwood, Nicholas J; Ballif, Bryan A; Ward, Gary E

    2010-01-15

    Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.

  6. Myosin VI: cellular functions and motor properties.

    PubMed Central

    Roberts, Rhys; Lister, Ida; Schmitz, Stephan; Walker, Matthew; Veigel, Claudia; Trinick, John; Buss, Folma; Kendrick-Jones, John

    2004-01-01

    Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed. PMID:15647169

  7. Myosin VI: cellular functions and motor properties.

    PubMed

    Roberts, Rhys; Lister, Ida; Schmitz, Stephan; Walker, Matthew; Veigel, Claudia; Trinick, John; Buss, Folma; Kendrick-Jones, John

    2004-12-29

    Myosin VI has been localized in membrane ruffles at the leading edge of cells, at the trans-Golgi network compartment of the Golgi complex and in clathrin-coated pits or vesicles, indicating that it functions in a wide variety of intracellular processes. Myosin VI moves along actin filaments towards their minus end, which is the opposite direction to all of the other myosins so far studied (to our knowledge), and is therefore thought to have unique properties and functions. To investigate the cellular roles of myosin VI, we identified various myosin VI binding partners and are currently characterizing their interactions within the cell. As an alternative approach, we have expressed and purified full-length myosin VI and studied its in vitro properties. Previous studies assumed that myosin VI was a dimer, but our biochemical, biophysical and electron microscopic studies reveal that myosin VI can exist as a stable monomer. We observed, using an optical tweezers force transducer, that monomeric myosin VI is a non-processive motor which, despite a relatively short lever arm, generates a large working stroke of 18 nm. Whether monomer and/or dimer forms of myosin VI exist in cells and their possible functions will be discussed. PMID:15647169

  8. Blebbistatin and blebbistatin-inactivated myosin II inhibit myosin II-independent processes in Dictyostelium

    PubMed Central

    Shu, Shi; Liu, Xiong; Korn, Edward D.

    2005-01-01

    Blebbistatin, a cell-permeable inhibitor of class-II myosins, was developed to provide a tool for studying the biologic roles of myosin II. Consistent with this use, we find that blebbistatin inhibits three myosin II-dependent processes in Dictyostelium (growth in suspension culture, capping of Con A receptors, and development to fruiting bodies) and does not inhibit growth on plates, which does not require myosin II. As expected, macropinocytosis (myosin I-dependent), contractile vacuole activity (myosin V-dependent), and phagocytosis (myosin VII-dependent), none of which requires myosin II, are not inhibited by blebbistatin in myosin II-null cells, but, unexpectedly, blebbistatin does inhibit macropinocytosis and phagocytosis by cells expressing myosin II. Expression of catalytically inactive myosin II in myosin II-null cells also inhibits macropinocytosis and phagocytosis. Both blebbistatin-inhibited myosin II and catalytically inactive myosin II form cytoplasmic aggregates, which may be why they inhibit myosin II-independent processes, but neither affects the distribution of actin filaments in vegetative cells or actin and myosin distribution in dividing or polarized cells. Blebbistatin also inhibits cell streaming and plaque expansion in myosin II-null cells. Our results are consistent with myosin II being the only Dictyostelium myosin that is inhibited by blebbistatin but also show that blebbistatin-inactivated myosin II inhibits some myosin II-independent processes and that blebbistatin inhibits other activities in the absence of myosin II. PMID:15671182

  9. Arabidopsis myosin XI: a motor rules the tracks.

    PubMed

    Cai, Chao; Henty-Ridilla, Jessica L; Szymanski, Daniel B; Staiger, Christopher J

    2014-11-01

    Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin filament arrays in epidermal cells. Furthermore, the overall actin dynamicity was significantly inhibited in the xi3KO mutant. Because cytoskeletal remodeling is contributed mainly by filament assembly/disassembly and translocation/buckling, we also examined the dynamic behavior of individual actin filaments. We found that the xi3KO mutant had significantly decreased actin turnover, with a 2-fold reduction in filament severing frequency. Moreover, quantitative analysis of filament shape change over time revealed that myosin XI generates the force for buckling and straightening of both single actin filaments and actin bundles. Thus, our data provide genetic evidence that three Arabidopsis class XI myosins contribute to actin remodeling by stimulating turnover and generating the force for filament shape change. PMID:25237128

  10. Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

    PubMed

    Sellers, James R; Wang, Fei; Chantler, Peter D

    2003-01-01

    Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

  11. Phospholipid-dependent regulation of the motor activity of myosin X.

    PubMed

    Umeki, Nobuhisa; Jung, Hyun Suk; Sakai, Tsuyoshi; Sato, Osamu; Ikebe, Reiko; Ikebe, Mitsuo

    2011-06-12

    Myosin X is involved in the reorganization of the actin cytoskeleton and protrusion of filopodia. Here we studied the molecular mechanism by which bovine myosin X is regulated. The globular tail domain inhibited the motor activity of myosin X in a Ca(2+)-independent manner. Structural analysis revealed that myosin X is monomeric and that the band 4.1-ezrin-radixin-moesin (FERM) and pleckstrin homology (PH) domains bind to the head intramolecularly, forming an inhibited conformation. Binding of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P(3)) to the PH domain reversed the tail-induced inhibition and induced the formation of myosin X dimers. Consistently, disruption of the binding of PtdIns(3,4,5)P(3) attenuated the translocation of myosin X to filopodial tips in cells. We propose the following mechanism: first, the tail inhibits the motor activity of myosin X by intramolecular head-tail interactions to form the folded conformation; second, phospholipid binding reverses the inhibition and disrupts the folded conformation, which induces dimer formation, thereby activating the mechanical and cargo transporter activity of myosin X.

  12. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  13. 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.

  14. Kinetic mechanism of the fastest motor protein, Chara myosin.

    PubMed

    Ito, Kohji; Ikebe, Mitsuo; Kashiyama, Taku; Mogami, Toshifumi; Kon, Takahide; Yamamoto, Keiichi

    2007-07-01

    Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin.motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s(-1), and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s(-1). From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a V(max) value of the actin-activated ATPase activity of 390 s(-1). The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.

  15. 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.

  16. Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor*

    PubMed Central

    Lu, Zekuan; Ma, Xiao-Nan; Zhang, Hai-Man; Ji, Huan-Hong; Ding, Hao; Zhang, Jie; Luo, Dan; Sun, Yujie; Li, Xiang-dong

    2014-01-01

    Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament. PMID:24825904

  17. Engineering controllable bidirectional molecular motors based on myosin

    NASA Astrophysics Data System (ADS)

    Chen, Lu; Nakamura, Muneaki; Schindler, Tony D.; Parker, David; Bryant, Zev

    2012-04-01

    Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells and have potential applications in molecular detection and diagnostic devices. Engineering molecular motors with controllable properties will allow selective perturbation of mechanical processes in living cells and provide optimized device components for tasks such as molecular sorting and directed assembly. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions and other signals. Here, we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies and guided by a structural model for the redirected power stroke of myosin VI, we have constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should make it possible to achieve spatiotemporal control over a range of motor properties including processivity, stride size and branchpoint turning.

  18. Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†

    PubMed Central

    Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2010-01-01

    Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

  19. Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

    SciTech Connect

    Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen; Kerekes, Ryan A; Gleason, Shaun Scott; Hatten, Mary E

    2009-01-01

    Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.

  20. Single-molecule studies of unconventional motor protein myosin VI

    NASA Astrophysics Data System (ADS)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  1. Electrostatic origin of the unidirectionality of walking myosin V motors.

    PubMed

    Mukherjee, Shayantani; Warshel, Arieh

    2013-10-22

    Understanding the basis for the action of myosin motors and related molecular machines requires a quantitative energy-based description of the overall functional cycle. Previous theoretical attempts to do so have provided interesting insights on parts of the cycle but could not generate a structure-based free energy landscape for the complete cycle of myosin. In particular, a nonphenomenological structure/energy-based understanding of the unidirectional motion is still missing. Here we use a coarse-grained model of myosin V and generate a structure-based free energy surface of the largest conformational change, namely the transition from the post- to prepowerstroke movement. We also couple the observed energetics of ligand binding/hydrolysis and product release to that of the conformational surface and reproduce the energetics of the complete mechanochemical cycle. It is found that the release in electrostatic free energy upon changing the conformation of the lever arm and the convertor domain from its post- to prepowerstroke state provides the necessary energy to bias the system towards the unidirectional movement of myosin V on the actin filament. The free energy change of 11 kcal is also in the range of ∼2-3 pN, which is consistent with the experimentally observed stalling force required to stop the motor completely on its track. The conformational-chemical coupling generating a successful powerstroke cycle is believed to be conserved among most members of the myosin family, thus highlighting the importance of the previously unknown role of electrostatics free energy in guiding the functional cycle in other actin-based myosin motors. PMID:24106304

  2. Does 2,3-butanedione monoxime inhibit nonmuscle myosin?

    PubMed

    Forer, Arthur; Fabian, Lacramioara

    2005-04-01

    BDM (2,3-butanedione monoxime) has been used extensively to inhibit nonmuscle myosin. However, recent articles raise the question of what BDM actually does, because of experiments in which BDM did not affect the actin-activated ATPase of nonmuscle myosins. We describe results that indicate that BDM indeed inhibits motility due to nonmuscle myosins: in many different cells BDM has the same effects as anti-actin agents and/or as other anti-myosin agents, and BDM slows or stops the sliding between actin filaments and myosin in vitro. We discuss how the two sets of apparently contradictory results might be resolved, and we suggest possible experiments that might clarify the contradictory interpretations. PMID:15868207

  3. Myosin-I molecular motors at a glance.

    PubMed

    McIntosh, Betsy B; Ostap, E Michael

    2016-07-15

    Myosin-I molecular motors are proposed to play various cellular roles related to membrane dynamics and trafficking. In this Cell Science at a Glance article and the accompanying poster, we review and illustrate the proposed cellular functions of metazoan myosin-I molecular motors by examining the structural, biochemical, mechanical and cell biological evidence for their proposed molecular roles. We highlight evidence for the roles of myosin-I isoforms in regulating membrane tension and actin architecture, powering plasma membrane and organelle deformation, participating in membrane trafficking, and functioning as a tension-sensitive dock or tether. Collectively, myosin-I motors have been implicated in increasingly complex cellular phenomena, yet how a single isoform accomplishes multiple types of molecular functions is still an active area of investigation. To fully understand the underlying physiology, it is now essential to piece together different approaches of biological investigation. This article will appeal to investigators who study immunology, metabolic diseases, endosomal trafficking, cell motility, cancer and kidney disease, and to those who are interested in how cellular membranes are coupled to the underlying actin cytoskeleton in a variety of different applications. PMID:27401928

  4. Optical trapping studies of acto-myosin motor proteins

    NASA Astrophysics Data System (ADS)

    Farrow, Rachel E.; Rosenthal, Peter B.; Mashanov, Gregory I.; Holder, Anthony A.; Molloy, Justin E.

    2007-09-01

    Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor proteins convert chemical energy to mechanical work. This paper focuses on studies of the acto-myosin motor system that is responsible for muscle contraction and a host of other cellular motilities. Myosin works by binding to filamentous actin, pulling and then releasing. Each cycle of interaction produces a few nanometres movement and a few piconewtons force. Individual interactions can be observed directly by holding an individual actin filament between two optically trapped microspheres and positioning it in the immediate vicinity of a single myosin motor. When the chemical fuel (adenosine triphosphate or ATP) is present the myosin undergoes repeated cycles of interaction with the actin filament producing square-wave like displacements and forces. Analysis of optical trapping data sets enables the size and timing of the molecular motions to be deduced.

  5. 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

    2012-06-01

    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.

  6. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  7. Catalytic strategy used by the myosin motor to hydrolyze ATP

    PubMed Central

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-01-01

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum–classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO42− state, but in the H2PγO4− state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3−) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes. PMID:25006262

  8. Changes in Mg2+ ion concentration and heavy chain phosphorylation regulate the motor activity of a class I myosin.

    PubMed

    Fujita-Becker, Setsuko; Dürrwang, Ulrike; Erent, Muriel; Clark, Richard J; Geeves, Michael A; Manstein, Dietmar J

    2005-02-18

    Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.

  9. Myosin VI as a transporter and an anchor: A model for kinetics of the motor under load

    NASA Astrophysics Data System (ADS)

    Chuan, Peiying; Spudich, James; Dunn, Alexander

    2010-03-01

    Myosin VI is an actin-based motor that is thought to function both as a transporter and an anchor in vivo. In an earlier study (Altman et al, Cell 2004), inhibition of myosin VI stepping kinetics by load applied using an optical trap was observed at saturating ATP and low ADP concentrations (< 2.5 μM). A simple mechanism whereby the rate of ADP binding increases exponentially with load was proposed. This model predicts that myosin VI functions primarily as an anchor at loads greater than ˜0.5 pN under physiological nucleotide conditions, which is potentially inconsistent with its roles in vivo. Here we present myosin VI stepping data taken at a variety of applied loads and ADP concentrations, and show that the Altman model only holds at low ADP concentrations. At higher, physiologically relevant ADP concentrations under load we observe dwell times that are an order of magnitude smaller than predicted by the Altman model. We present a modified model in which applied load alters the equilibrium between two myosin VI states with different nucleotide affinities. This new kinetic scheme accurately describes myosin VI behavior at various nucleotide conditions under a large range of loads, and explains how the motor is able to carry out its roles in vivo, both as a force-generating transporter and as an anchor.

  10. Life without double-headed non-muscle myosin II motor proteins

    NASA Astrophysics Data System (ADS)

    Betapudi, Venkaiah

    2014-07-01

    Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC) belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  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. Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.

    PubMed

    Diensthuber, Ralph P; Tominaga, Motoki; Preller, Matthias; Hartmann, Falk K; Orii, Hidefumi; Chizhov, Igor; Oiwa, Kazuhiro; Tsiavaliaris, Georgios

    2015-01-01

    The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins. PMID:25326536

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

    PubMed

    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

  14. 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

  15. 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.

  16. 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.

  17. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments.

    PubMed

    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. PMID:26149240

  18. Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

    PubMed

    Batters, Christopher; Woodall, Katy A; Toseland, Christopher P; Hundschell, Christian; Veigel, Claudia

    2012-08-10

    The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle. PMID:22718767

  19. Strain Mediated Adaptation Is Key for Myosin Mechanochemistry: Discovering General Rules for Motor Activity.

    PubMed

    Jana, Biman; Onuchic, José N

    2016-08-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. PMID:27494025

  20. Strain Mediated Adaptation Is Key for Myosin Mechanochemistry: Discovering General Rules for Motor Activity

    PubMed Central

    Jana, Biman; Onuchic, José N.

    2016-01-01

    A structure-based model of myosin motor is built in the same spirit of our early work for kinesin-1 and Ncd towards physical understanding of its mechanochemical cycle. We find a structural adaptation of the motor head domain in post-powerstroke state that signals faster ADP release from it compared to the same from the motor head in the pre-powerstroke state. For dimeric myosin, an additional forward strain on the trailing head, originating from the postponed powerstroke state of the leading head in the waiting state of myosin, further increases the rate of ADP release. This coordination between the two heads is the essence of the processivity of the cycle. Our model provides a structural description of the powerstroke step of the cycle as an allosteric transition of the converter domain in response to the Pi release. Additionally, the variation in structural elements peripheral to catalytic motor domain is the deciding factor behind diverse directionalities of myosin motors (myosin V & VI). Finally, we observe that there are general rules for functional molecular motors across the different families. Allosteric structural adaptation of the catalytic motor head in different nucleotide states is crucial for mechanochemistry. Strain-mediated coordination between motor heads is essential for processivity and the variation of peripheral structural elements is essential for their diverse functionalities. PMID:27494025

  1. Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

    PubMed Central

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

    2014-01-01

    Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm2), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track. PMID:25201964

  2. Crystal structure of the rigor-like human non-muscle myosin-2 motor domain.

    PubMed

    Münnich, Stefan; Pathan-Chhatbar, Salma; Manstein, Dietmar J

    2014-12-20

    We determined the crystal structure of the motor domain of human non-muscle myosin 2B (NM-2B) in a nucleotide-free state and at a resolution of 2.8 Å. The structure shows the motor domain with an open active site and the large cleft that divides the 50 kDa domain in a closed state. Compared to other rigor-like myosin motor domain structures, our structure shows subtle but significant conformational changes in regions important for actin binding and mechanochemical coupling. Moreover, our crystal structure helps to rationalize the impact of myosin, heavy chain 9 (MYH9)-related disease mutations Arg709Cys and Arg709His on the kinetic and functional properties of NM-2B and of the closely related non-muscle myosin 2A (NM-2A). PMID:25451231

  3. Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin.

    PubMed

    Ito, Kohji; Yamaguchi, Yukie; Yanase, Kenji; Ichikawa, Yousuke; Yamamoto, Keiichi

    2009-12-22

    Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected V(max) and K(app) of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected K(app) and the velocity, although it did not affect V(max). Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.

  4. Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

    PubMed Central

    Uyeda, Taro Q. P.; Iwadate, Yoshiaki; Umeki, Nobuhisa; Nagasaki, Akira; Yumura, Shigehiko

    2011-01-01

    To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli. PMID:22022566

  5. Various Themes of Myosin Regulation.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2016-05-01

    Members of the myosin superfamily are actin-based molecular motors that are indispensable for cellular homeostasis. The vast functional and structural diversity of myosins accounts for the variety and complexity of the underlying allosteric regulatory mechanisms that determine the activation or inhibition of myosin motor activity and enable precise timing and spatial aspects of myosin function at the cellular level. This review focuses on the molecular basis of posttranslational regulation of eukaryotic myosins from different classes across species by allosteric intrinsic and extrinsic effectors. First, we highlight the impact of heavy and light chain phosphorylation. Second, we outline intramolecular regulatory mechanisms such as autoinhibition and subsequent activation. Third, we discuss diverse extramolecular allosteric mechanisms ranging from actin-linked regulatory mechanisms to myosin:cargo interactions. At last, we briefly outline the allosteric regulation of myosins with synthetic compounds.

  6. 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.

  7. The motility of Chara corallina myosin was inhibited reversibly by 2,3-butanedione monoxime (BDM).

    PubMed

    Funaki, Keisuke; Nagata, Ayumi; Akimoto, Youka; Shimada, Kiyo; Ito, Kohji; Yamamoto, Keiichi

    2004-09-01

    We studied the effects of 2,3-butanedione monoxime (BDM) on the cytoplasmic streaming of Chara corallina and on the motility of myosin prepared from the same plant to examine whether this reagent really affects the plant class XI myosin. It was found that BDM inhibited both cytoplasmic streaming and the motility of myosin at a very similar concentration range (10-100 mM). BDM introduced directly into tonoplast-free cells also inhibited cytoplasmic streaming. These results suggested that effect of BDM on cytoplasmic streaming was exerted through myosin and not through ion channels at least in Chara corallina, though a very high concentration of BDM was required.

  8. The non-linear elasticity of the muscle sarcomere and the compliance of myosin motors

    PubMed Central

    Fusi, Luca; Brunello, Elisabetta; Reconditi, Massimo; Piazzesi, Gabriella; Lombardi, Vincenzo

    2014-01-01

    Force in striated muscle is due to attachment of the heads of the myosin, the molecular motors extending from the myosin filament, to the actin filament in each half-sarcomere, the functional unit where myosin motors act in parallel. Mechanical and X-ray structural evidence indicates that at the plateau of isometric contraction (force T0), less than half of the elastic strain of the half-sarcomere is due to the strain in the array of myosin motors (s), with the remainder being accounted for by the compliance of filaments acting as linear elastic elements in series with the motor array. Early during the development of isometric force, however, the half-sarcomere compliance has been found to be less than that expected from the linear elastic model assumed above, and this non-linearity may affect the estimate of s. This question is investigated here by applying nanometre–microsecond-resolution mechanics to single intact fibres from frog skeletal muscle at 4°C, to record the mechanical properties of the half-sarcomere throughout the development of force in isometric contraction. The results are interpreted with mechanical models to estimate the compliance of the myosin motors. Our conclusions are as follows: (i) early during the development of an isometric tetanus, an elastic element is present in parallel with the myosin motors, with a compliance of ∼200 nm MPa−1 (∼20 times larger than the compliance of the motor array at T0); and (ii) during isometric contraction, s is 1.66 ± 0.05 nm, which is not significantly different from the value estimated with the linear elastic model. PMID:24344166

  9. A small molecule species specifically inhibits Fusarium myosin I.

    PubMed

    Zhang, Chengqi; Chen, Yun; Yin, Yanni; Ji, Huan-Hong; Shim, Won-Bo; Hou, Yiping; Zhou, Mingguo; Li, Xiang-Dong; Ma, Zhonghua

    2015-08-01

    Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease of cereal crops worldwide. Recently, a novel fungicide JS399-19 has been launched into the marketplace to manage FHB. It is compelling that JS399-19 shows highly inhibitory activity towards some Fusarium species, but not to other fungi, indicating that it is an environmentally compatible fungicide. To explore the mode of action of this species-specific compound, we conducted a whole-genome transcript profiling together with genetic and biochemical assays, and discovered that JS399-19 targets the myosin I of F. graminearum (FgMyo1). FgMyo1 is essential for F. graminearum growth. A point mutation S217L or E420K in FgMyo1 is responsible for F. graminearum resistance to JS399-19. In addition, transformation of F. graminearum with the myosin I gene of Magnaporthe grisea, the causal agent of rice blast, also led to JS399-19 resistance. JS399-19 strongly inhibits the ATPase activity of the wild-type FgMyo1, but not the mutated FgMyo1(S217L/E420K) . These results provide us a new insight into the design of species-specific antifungal compounds. Furthermore, our strategy can be applied to identify novel drug targets in various pathogenic organisms.

  10. 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

  11. 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

    2016-01-01

    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. PMID:27623148

  12. Myosin II motor activity in the lateral amygdala is required for fear memory consolidation.

    PubMed

    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 recently shown that these molecular machines mobilize F-actin in response to synaptic stimulation and learning in the hippocampus. In this study, we report that Myosin II motors in the rat lateral amygdala (LA) are essential for fear memory formation. Pretraining infusions of the Myosin II inhibitor, blebbistatin (blebb), disrupted long term memory, while short term memory was unaffected. Interestingly, both post-training and pretesting infusions had no effect on memory formation, indicating that Myosin II motors operate during or shortly after learning to promote memory consolidation. These data support the idea that Myosin II motor-force generation is a general mechanism that supports memory consolidation in the mammalian CNS.

  13. 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.

  14. Insights into the Chemomechanical Coupling of the Myosin Motor from Simulation of Its ATP Hydrolysis Mechanism

    SciTech Connect

    Schwarzl, S.M.; Smith, Jeremy C; Fischer, S.

    2006-03-01

    The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP{circle_dot}Pi state of nucleotide-bound myosin.

  15. The myofilament elasticity and its effect on kinetics of force generation by the myosin motor.

    PubMed

    Piazzesi, Gabriella; Dolfi, Mario; Brunello, Elisabetta; Fusi, Luca; Reconditi, Massimo; Bianco, Pasquale; Linari, Marco; Lombardi, Vincenzo

    2014-06-15

    The half-sarcomere is the functional unit of striated muscle, in which, according to a "linear" mechanical model, myosin motors are parallel force generators with an average strain s acting between the opposing myosin and actin filaments that behave as a series elastic element with compliance Cf. Thus the definition of the mechanism of force generation by myosin motors in muscle requires integration of the crystallographic model of the working stroke with the mechanical constraints provided by the organization of motors in the half-sarcomere. The relation between half-sarcomere compliance and force (Chs-T) during the development of isometric contraction deviates, at low forces, from that predicted by the linear model, indicating the presence of an elastic element in parallel with the myosin motors, which may influence the estimate of s. A working stroke model, kinetically constrained by the early phase of the isotonic velocity transient following a force step, predicts that the rate of quick force recovery following a length step is reduced to the observed value by a Cf of 12.6nm/MPa. With this value of Cf, the fit of Chs-T relation during the isometric force rise gives s=1.8-1.9nm, similar to the values estimated using the linear model. PMID:24631572

  16. 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…

  17. Aberrant post-translational modifications compromise human myosin motor function in old age.

    PubMed

    Li, Meishan; Ogilvie, Hannah; Ochala, Julien; Artemenko, Konstantin; Iwamoto, Hiroyuki; Yagi, Naoto; Bergquist, Jonas; Larsson, Lars

    2015-04-01

    Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

  18. Molecular genetics of myosin motors in Arabidopsis. Final report, July 1, 1992--June 30, 1996

    SciTech Connect

    Schiefelbein, J.

    1997-02-01

    The normal growth and development of plant cells depends on the precise organization and distribution of the cellular contents. The basic goal of this investigation was to define a group of the molecules that are involved in organizing and transporting plant cell components. Based largely on studies of animal and fungal cells, one of the molecules thought to be involved in intracellular trafficking in plants is the actin-based motor protein myosin. Therefore, the major aim of this study was to isolate and analyze plant genes encoding myosin proteins. The plant of choice for these experiments was Arabidopsis thaliana, which offers numerous advantages for molecular genetics research.

  19. Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

    PubMed

    Brzeska, H; Kulesza-Lipka, D; Korn, E D

    1992-11-25

    The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities. PMID:1331103

  20. Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility

    PubMed Central

    Hanley, Peter J.; Xu, Yan; Kronlage, Moritz; Grobe, Kay; Schön, Peter; Song, Jian; Sorokin, Lydia; Schwab, Albrecht; Bähler, Martin

    2010-01-01

    Directional motility is a fundamental function of immune cells, which are recruited to sites of pathogen invasion or tissue damage by chemoattractant signals. To move, cells need to generate lamellipodial membrane protrusions at the front and retract the trailing end. These elementary events are initiated by Rho-family GTPases, which cycle between active GTP-bound and inactive GDP-bound states. How the activity of these “molecular switches” is spatially coordinated is only beginning to be understood. Here, we show that myosin IXb (Myo9b), a Rho GTPase-activating protein (RhoGAP) expressed in immune cells, is essential for coordinating the activity of Rho. We generated Myo9b-deficient mice and show that Myo9b−/− macrophages have strikingly defective spreading and polarization. Furthermore, Myo9b−/− macrophages fail to generate lamellipodia in response to a chemoattractant, and migration in a chemotactic gradient is severely impaired. Inhibition of Rho rescues the Myo9b−/− phenotype, but impairs tail retraction. We also found that Myo9b is important in vivo. Chemoattractant-induced monocyte recruitment to the peritoneal cavity is substantially reduced in Myo9b−/− mice. Thus, we identify the “motorized Rho inhibitor” Myo9b as a key molecular component required for spatially coordinated cell shape changes and motility. PMID:20566876

  1. The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor

    SciTech Connect

    Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2006-12-01

    Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by {approx}60 degrees. This recovery stroke is coupled to the activation of myosin's ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a see-saw motion of the relay helix, followed by a piston/seesaw motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery stroke by using Principal Component Analysis. This reveals that the only principal motions of these two helices that make a large amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions.

  2. SH1 (cysteine 717) of smooth muscle myosin: its role in motor function.

    PubMed

    Kojima, S; Fujiwara, K; Onishi, H

    1999-09-01

    To determine if a thiol group called SH1 has an important role in myosin's motor function, we made a mutant heavy meromyosin (HMM) without the thiol group and analyzed its properties. In chicken gizzard myosin, SH1 is located on the cysteine residue at position 717. By using genetic engineering techniques, this cysteine was substituted with threonine in chicken gizzard HMM, and that mutant HMM and unmutated HMM were expressed in biochemical quantities using a baculovirus system. The basal EDTA-, Ca(2+)-, and Mg(2+)-ATPase activities of the mutant were similar to those of HMM whose SH1 was modified by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS). However, while the chemically modified HMM lost the function of the light chain phosphorylation-dependent regulation of the actin-activated ATPase activity, the mutant HMM exhibited the normal light chain-regulated actin-activated ATPase activity. Using an in vitro motility assay system, we found that the IAEDANS-modified HMM was unable to propel actin filaments but that the mutant HMM was able to move actin filaments in a manner indistinguishable from filament sliding generated by unmutated HMM. These results indicate that SH1 itself is not essential for the motor function of myosin and suggest that various effects observed with HMM modified by thiol reagents such as IAEDANS are caused by the bulkiness of the attached probes, which interferes with the swinging motion generated during ATP hydrolysis.

  3. Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

    PubMed Central

    Logvinova, Daria S.; Markov, Denis I.; Nikolaeva, Olga P.; Sluchanko, Nikolai N.; Ushakov, Dmitry S.; Levitsky, Dmitrii I.

    2015-01-01

    Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF4- and S1-ADP-BeFx significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle. PMID:26356744

  4. Neural crest specification by inhibition of the ROCK/Myosin II pathway

    PubMed Central

    Kim, Kyeongmi; Ossipova, Olga; Sokol, Sergei Y.

    2015-01-01

    Neural crest is a population of multipotent progenitor cells that form at the border of neural and non-neural ectoderm in vertebrate embryos, and undergo epithelialmesenchymal transition and migration. According to the traditional view, the neural crest is specified in early embryos by signaling molecules including BMP, FGF and Wnt proteins. Here we identify a novel signaling pathway leading to neural crest specification, which involves Rho-associated kinase (ROCK) and its downstream target non-muscle Myosin II. We show that ROCK inhibitors promote differentiation of human embryonic stem cells into neural crest-like progenitors (NCPs) that are characterized by specific molecular markers and ability to differentiate into multiple cell types, including neurons, chondrocytes, osteocytes and smooth muscle cells. Moreover, inhibition of Myosin II was sufficient for generating NCPs at high efficiency. Whereas Myosin II has been previously implicated in the self-renewal and survival of human pluripotent ES cells, we demonstrate its role in neural crest development during ES cell differentiation. Inhibition of this pathway in Xenopus embryos expanded neural crest in vivo, further indicating that neural crest specification is controlled by ROCK-dependent Myosin II activity. We propose that changes in cell morphology in response to ROCK and Myosin II inhibition initiate mechanical signaling leading to neural crest fates. PMID:25346532

  5. Small-molecule inhibitors of myosin proteins

    PubMed Central

    Bond, Lisa M; Tumbarello, David A; Kendrick-Jones, John; Buss, Folma

    2014-01-01

    Advances in screening and computational methods have enhanced recent efforts to discover/design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design. PMID:23256812

  6. Nucleotide-Dependent Shape Changes in the Reverse Direction Motor, Myosin VI

    PubMed Central

    Song, Chun Feng; Sader, Kasim; White, Howard; Kendrick-Jones, John; Trinick, John

    2010-01-01

    We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca2+). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO4, which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix. PMID:21081082

  7. Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity.

    PubMed

    Ito, Kohji; Kashiyama, Taku; Shimada, Kiyo; Yamaguchi, Akira; Awata, Jun ya; Hachikubo, You; Manstein, Dietmar J; Yamamoto, Keiichi

    2003-12-26

    The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.

  8. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    NASA Astrophysics Data System (ADS)

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.

    2015-08-01

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  9. Small molecule-mediated refolding and activation of myosin motor function

    PubMed Central

    Radke, Michael B; Taft, Manuel H; Stapel, Britta; Hilfiker-Kleiner, Denise; Preller, Matthias; Manstein, Dietmar J

    2014-01-01

    The small molecule EMD 57033 has been shown to stimulate the actomyosin ATPase activity and contractility of myofilaments. Here, we show that EMD 57033 binds to an allosteric pocket in the myosin motor domain. EMD 57033-binding protects myosin against heat stress and thermal denaturation. In the presence of EMD 57033, ATP hydrolysis, coupling between actin and nucleotide binding sites, and actin affinity in the presence of ATP are increased more than 10-fold. Addition of EMD 57033 to heat-inactivated β-cardiac myosin is followed by refolding and reactivation of ATPase and motile activities. In heat-stressed cardiomyocytes expression of the stress-marker atrial natriuretic peptide is suppressed by EMD 57033. Thus, EMD 57033 displays a much wider spectrum of activities than those previously associated with small, drug-like compounds. Allosteric effectors that mediate refolding and enhance enzymatic function have the potential to improve the treatment of heart failure, myopathies, and protein misfolding diseases. DOI: http://dx.doi.org/10.7554/eLife.01603.001 PMID:24520162

  10. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity.

    PubMed

    Winkelmann, Donald A; Forgacs, Eva; Miller, Matthew T; Stock, Ann M

    2015-08-06

    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  11. The principal motions involved in the coupling mechanism of the recovery stroke of the myosin motor.

    SciTech Connect

    Mesentean, Sidonia; Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-03-01

    Muscle contraction is driven by a cycle of conformational changes in the myosin II head. After myosin binds ATP and releases from the actin fibril, myosin prepares for the next power stroke by rotating back the converter domain that carries the lever arm by 60{sup o}. This recovery stroke is coupled to the activation of myosin ATPase by a mechanism that is essential for an efficient motor cycle. The mechanics of this coupling have been proposed to occur via two distinct and successive motions of the two helices that hold the converter domain: in a first phase a seesaw motion of the relay helix, followed by a piston-like motion of the SH1 helix in a second phase. To test this model, we have determined the principal motions of these structural elements during equilibrium molecular dynamics simulations of the crystallographic end states of the recovery-stroke by using principal component analysis. This reveals that the only principal motions of these two helices that make a large-amplitude contribution towards the conformational change of the recovery stroke are indeed the predicted seesaw and piston motions. Moreover, the results demonstrate that the seesaw motion of the relay helix dominates in the dynamics of the pre-recovery stroke structure, but not in the dynamics of the post-recovery stroke structure, and vice versa for the piston motion of the SH1 helix. This is consistent with the order of the proposed two-phase model for the coupling mechanism of the recovery stroke. Molecular movies of these principal motions are available at http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer.

  12. Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity.

    PubMed

    Smith, Clayton; Dolat, Lee; Angelis, Dimitrios; Forgacs, Eva; Spiliotis, Elias T; Galkin, Vitold E

    2015-10-01

    Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.

  13. The structural coupling between ATPase activation and recovery stroke in the myosin II motor

    SciTech Connect

    Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-07-01

    Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

  14. Force and number of myosin motors during muscle shortening and the coupling with the release of the ATP hydrolysis products

    PubMed Central

    Caremani, Marco; Melli, Luca; Dolfi, Mario; Lombardi, Vincenzo; Linari, Marco

    2015-01-01

    The chemo-mechanical cycle of the myosin II–actin reaction in situ has been investigated in Ca2+-activated skinned fibres from rabbit psoas, by determining the number and strain (s) of myosin motors interacting during steady shortening at different velocities (V) and the effect of raising inorganic phosphate (Pi) concentration. It was found that in control conditions (no added Pi), shortening at V ≤ 350 nm s–1 per half-sarcomere, corresponding to force (T) greater than half the isometric force (T0), decreases the number of myosin motors in proportion to the reduction of T, so that s remains practically constant and similar to the T0 value independent of V. At higher V the number of motors decreases less than in proportion to T, so that s progressively decreases. Raising Pi concentration by 10 mm, which reduces T0 and the number of motors by 40–50%, does not influence the dependence on V of number and strain. A model simulation of the myosin–actin reaction in which the structural transitions responsible for the myosin working stroke and the release of the hydrolysis products are orthogonal explains the results assuming that Pi and then ADP are released with rates that increase as the motor progresses through the working stroke. The rate of ADP release from the conformation at the end of the working stroke is also strain-sensitive, further increasing by one order of magnitude within a few nanometres of negative strain. These results provide the molecular explanation of the relation between the rate of energy liberation and the load during muscle contraction. Key points Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin–actin interactions increases with the increase in shortening velocity. We used fast half-sarcomere mechanics on skinned muscle fibres to

  15. Harmonic force spectroscopy reveals a force-velocity curve from a single human beta cardiac myosin motor

    NASA Astrophysics Data System (ADS)

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian; Mortensen, Kim; Flyvbjerg, Henrik; Spudich, James

    2014-03-01

    A muscle contracts rapidly under low load, but slowly under high load. Its molecular mechanisms remain to be elucidated, however. During contraction, myosins in thick filaments interact with actin in thin filaments in the sarcomere, cycling between a strongly bound (force producing) state and a weakly bound (relaxed) state. Huxley et al. have previously proposed that the transition from the strong to the weak interaction can be modulated by a load. We use a new method we call ``harmonic force spectroscopy'' to extract a load-velocity curve from a single human beta cardiac myosin II motor. With a dual-beam optical trap, we hold an actin dumbbell over a myosin molecule anchored to the microscope stage that oscillates sinusoidally. Upon binding, the motor experiences an oscillatory load with a mean that is directed forward or backward, depending on binding location We find that the bound time at saturating [ATP] is exponentially correlated with the mean load, which is explained by Arrhenius transition theory. With a stroke size measurement, we obtained a load-velocity curve from a single myosin. We compare the curves for wild-type motors with mutants that cause hypertrophic cardiomyopathies, to understand the effects on the contractile cycle

  16. 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.

  17. A Millennial Myosin Census

    PubMed Central

    Berg, Jonathan S.; Powell, Bradford C.; Cheney, Richard E.

    2001-01-01

    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. PMID:11294886

  18. Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle.

    PubMed

    Trivedi, Darshan V; Muretta, Joseph M; Swenson, Anja M; Thomas, David D; Yengo, Christopher M

    2013-07-01

    We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

  19. Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance.

    PubMed

    Jacot, Damien; Daher, Wassim; Soldati-Favre, Dominique

    2013-06-12

    Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so-called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin-based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.

  20. Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis

    PubMed Central

    Spudich, James A.; Sivaramakrishnan, Sivaraj

    2010-01-01

    The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual ~ 180° lever arm swing and achieves its large step size using special structural features in its tail domain. PMID:20094053

  1. Response inhibition in motor conversion disorder.

    PubMed

    Voon, Valerie; Ekanayake, Vindhya; Wiggs, Edythe; Kranick, Sarah; Ameli, Rezvan; Harrison, Neil A; Hallett, Mark

    2013-05-01

    Conversion disorders (CDs) are unexplained neurological symptoms presumed to be related to a psychological issue. Studies focusing on conversion paralysis have suggested potential impairments in motor initiation or execution. Here we studied CD patients with aberrant or excessive motor movements and focused on motor response inhibition. We also assessed cognitive measures in multiple domains. We compared 30 CD patients and 30 age-, sex-, and education-matched healthy volunteers on a motor response inhibition task (go/no go), along with verbal motor response inhibition (color-word interference) and measures of attention, sustained attention, processing speed, language, memory, visuospatial processing, and executive function including planning and verbal fluency. CD patients had greater impairments in commission errors on the go/no go task (P < .001) compared with healthy volunteers, which remained significant after Bonferroni correction for multiple comparisons and after controlling for attention, sustained attention, depression, and anxiety. There were no significant differences in other cognitive measures. We highlight a specific deficit in motor response inhibition that may play a role in impaired inhibition of unwanted movement such as the excessive and aberrant movements seen in motor conversion. Patients with nonepileptic seizures, a different form of conversion disorder, are commonly reported to have lower IQ and multiple cognitive deficits. Our results point toward potential differences between conversion disorder subgroups. © 2013 Movement Disorder Society.

  2. CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

    PubMed

    Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

    2015-01-01

    Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that calix[4]arene C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus in vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

  3. Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

    PubMed Central

    Ebrahim, Seham; Avenarius, Matthew R.; Grati, M'hamed; Krey, Jocelyn F.; Windsor, Alanna M.; Sousa, Aurea D.; Ballesteros, Angela; Cui, Runjia; Millis, Bryan A.; Salles, Felipe T.; Baird, Michelle A.; Davidson, Michael W.; Jones, Sherri M.; Choi, Dongseok; Dong, Lijin; Raval, Manmeet H.; Yengo, Christopher M.; Barr-Gillespie, Peter G.; Kachar, Bechara

    2016-01-01

    Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. PMID:26926603

  4. Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.

    PubMed

    Ebrahim, Seham; Avenarius, Matthew R; Grati, M'hamed; Krey, Jocelyn F; Windsor, Alanna M; Sousa, Aurea D; Ballesteros, Angela; Cui, Runjia; Millis, Bryan A; Salles, Felipe T; Baird, Michelle A; Davidson, Michael W; Jones, Sherri M; Choi, Dongseok; Dong, Lijin; Raval, Manmeet H; Yengo, Christopher M; Barr-Gillespie, Peter G; Kachar, Bechara

    2016-01-01

    Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell. PMID:26926603

  5. 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

  6. Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

    PubMed Central

    Malmqvist, Ulf; Trybus, Kathleen M.; Yagi, Shinobu; Carmichael, Jeff; Fay, Fredric S.

    1997-01-01

    A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle. PMID:9207148

  7. The Motor Protein Myosin-X Transports VE-Cadherin along Filopodia To Allow the Formation of Early Endothelial Cell-Cell Contacts▿ †

    PubMed Central

    Almagro, Sébastien; Durmort, Claire; Chervin-Pétinot, Adeline; Heyraud, Stephanie; Dubois, Mathilde; Lambert, Olivier; Maillefaud, Camille; Hewat, Elizabeth; Schaal, Jean Patrick; Huber, Philippe; Gulino-Debrac, Danielle

    2010-01-01

    Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VE-cadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings. PMID:20123970

  8. Myosin II Activity Softens Cells in Suspension.

    PubMed

    Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-04-21

    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

  9. 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

  10. Myosin II Activity Softens Cells in Suspension.

    PubMed

    Chan, Chii J; Ekpenyong, Andrew E; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-04-21

    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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24591646

  12. 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.

  13. 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

  14. The Autophagy Receptor TAX1BP1 and the Molecular Motor Myosin VI Are Required for Clearance of Salmonella Typhimurium by Autophagy.

    PubMed

    Tumbarello, David A; Manna, Paul T; Allen, Mark; Bycroft, Mark; Arden, Susan D; Kendrick-Jones, John; Buss, Folma

    2015-10-01

    Autophagy plays a key role during Salmonella infection, by eliminating these pathogens following escape into the cytosol. In this process, selective autophagy receptors, including the myosin VI adaptor proteins optineurin and NDP52, have been shown to recognize cytosolic pathogens. Here, we demonstrate that myosin VI and TAX1BP1 are recruited to ubiquitylated Salmonella and play a key role in xenophagy. The absence of TAX1BP1 causes an accumulation of ubiquitin-positive Salmonella, whereas loss of myosin VI leads to an increase in ubiquitylated and LC3-positive bacteria. Our structural studies demonstrate that the ubiquitin-binding site of TAX1BP1 overlaps with the myosin VI binding site and point mutations in the TAX1BP1 zinc finger domains that affect ubiquitin binding also ablate binding to myosin VI. This mutually exclusive binding and the association of TAX1BP1 with LC3 on the outer limiting membrane of autophagosomes may suggest a molecular mechanism for recruitment of this motor to autophagosomes. The predominant role of TAX1BP1, a paralogue of NDP52, in xenophagy is supported by our evolutionary analysis, which demonstrates that functionally intact NDP52 is missing in Xenopus and mice, whereas TAX1BP1 is expressed in all vertebrates analysed. In summary, this work highlights the importance of TAX1BP1 as a novel autophagy receptor in myosin VI-mediated xenophagy. Our study identifies essential new machinery for the autophagy-dependent clearance of Salmonella typhimurium and suggests modulation of myosin VI motor activity as a potential therapeutic target in cellular immunity.

  15. Surround inhibition in motor execution and motor imagery.

    PubMed

    Aoyama, Toshiyuki; Kaneko, Fuminari; Ohashi, Yukari; Nagata, Hiroshi

    2016-08-26

    Surround inhibition (SI) is a neural mechanism to focus neuronal activity and facilitate selective motor execution (ME). The aim of the present study was to investigate whether SI is also generated during motor imagery (MI). Furthermore, we investigated whether the extent of SI during MI depends on the strength of SI during ME and/or vividness of MI. The extent of SI was examined during MI and ME of index finger flexion. Transcranial magnetic stimulation was applied at rest, during initiation of the movement (phasic phase) and during tonic muscle contraction of the index finger flexors. Motor evoked potentials (MEPs) were recorded from a surround muscle, abductor digiti minimi (ADM) and a synergistic muscle, the first dorsal interosseous muscle. The amplitude of ADM MEP was reduced during the phasic phase, which indicates that SI occurred during ME. In seven of 14 subjects, SI was also observed during MI, although this effect was not significant. There was a moderate correlation between the extent of SI during ME and MI. Furthermore, good imagers who experienced vivid MI during the MI task showed stronger SI than poor imagers. These results indicate that common neural substrates involved in SI during ME are at least in part recruited during MI. In clinical situations, the therapeutic use of MI to generate vivid MI may be one of effective tool to develop the strength of SI, which facilitate selective execution of desired movements. PMID:27418120

  16. 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.

  17. Engineered kinesin motor proteins amenable to small-molecule inhibition.

    PubMed

    Engelke, Martin F; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T Lynne; Soppina, Pushpanjali; Hancock, William O; Gelfand, Vladimir I; Verhey, Kristen J

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  18. Engineered kinesin motor proteins amenable to small-molecule inhibition

    PubMed Central

    Engelke, Martin F.; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

    2016-01-01

    The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

  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. Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

    PubMed Central

    Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

    1989-01-01

    Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

  1. Remote control of myosin and kinesin motors using light-activated gearshifting.

    PubMed

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C; Schindler, Tony D; Nogales, Eva; Bryant, Zev

    2014-09-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

  2. Remote control of myosin and kinesin motors using light-activated gearshifting.

    PubMed

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C; Schindler, Tony D; Nogales, Eva; Bryant, Zev

    2014-09-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport. PMID:25086603

  3. Remote control of myosin and kinesin motors using light-activated gearshifting

    PubMed Central

    Nakamura, Muneaki; Chen, Lu; Howes, Stuart C.; Schindler, Tony D.; Nogales, Eva

    2015-01-01

    Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells1,2. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems3,4. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears—speed up, slow down or switch directions—when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport. PMID:25086603

  4. Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

    PubMed Central

    Durrbach, A; Collins, K; Matsudaira, P; Louvard, D; Coudrier, E

    1996-01-01

    Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692943

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

    PubMed

    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

  6. 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

  7. Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum

    PubMed Central

    Swärd, Karl; Dreja, Karl; Susnjar, Marija; Hellstrand, Per; Hartshorne, David J; Walsh, Michael P

    2000-01-01

    Ca2+ sensitization of smooth muscle contraction involves the small GTPase RhoA, inhibition of myosin light chain phosphatase (MLCP) and enhanced myosin regulatory light chain (LC20) phosphorylation. A potential effector of RhoA is Rho-associated kinase (ROK).The role of ROK in Ca2+ sensitization was investigated in guinea-pig ileum.Contraction of permeabilized muscle strips induced by GTPγS at pCa 6.5 was inhibited by the kinase inhibitors Y-27632, HA1077 and H-7 with IC50 values that correlated with the known Ki values for inhibition of ROK. GTPγS also increased LC20 phosphorylation and this was prevented by HA1077. Contraction and LC20 phosphorylation elicited at pCa 5.75 were, however, unaffected by HA1077.Pre-treatment of intact tissue strips with HA1077 abolished the tonic component of carbachol-induced contraction and the sustained elevation of LC20 phosphorylation, but had no effect on the transient or sustained increase in [Ca2+]i induced by carbachol.LC20 phosphorylation and contraction dynamics suggest that the ROK-mediated increase in LC20 phosphorylation is due to MLCP inhibition, not myosin light chain kinase activation.In the absence of Ca2+, GTPγS stimulated 35S incorporation from [35S]ATPγS into the myosin targeting subunit of MLCP (MYPT). The enhanced thiophosphorylation was inhibited by HA1077. No thiophosphorylation of LC20 was detected.These results indicate that ROK mediates agonist-induced increases in myosin phosphorylation and force by inhibiting MLCP activity through phosphorylation of MYPT. Under Ca2+-free conditions, ROK does not appear to phosphorylate LC20in situ, in contrast to its ability to phosphorylate myosin in vitro. In particular, ROK activation is essential for the tonic phase of agonist-induced contraction. PMID:10618150

  8. Vaccinia virus F11 promotes viral spread by acting as a PDZ-containing scaffolding protein to bind myosin-9A and inhibit RhoA signaling.

    PubMed

    Handa, Yutaka; Durkin, Charlotte H; Dodding, Mark P; Way, Michael

    2013-07-17

    The vaccinia F11 protein promotes viral spread by modulating the cortical actin cytoskeleton by inhibiting RhoA signaling via an unknown mechanism. PDZ domains are widely conserved protein interaction modules whose occurrence in viral proteins is unprecedented. We found that F11 contains a central PDZ-like domain that is required to downregulate RhoA signaling and enhance viral spread. The PDZ-like domain interacts with the PDZ binding motif of the Rho GTPase-activating protein (GAP) Myosin-9A. In the absence of Myosin-9A, RhoA signaling is not inhibited, resulting in fewer actin tails and reduced virus release concomitant with less viral spread. The loss of Myosin-9A GAP activity or its ability to bind F11 also reduces actin tail formation. Furthermore, the ability of Myosin-9A to promote viral spread depends on F11 binding RhoA. Thus, F11 acts as a functional PDZ-containing scaffolding protein to inhibit RhoA signaling by binding Myosin-9A.

  9. Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress

    PubMed Central

    Gaji, Rajshekhar Y.; Johnson, Derrick E.; Treeck, Moritz; Wang, Mu; Hudmon, Andy; Arrizabalaga, Gustavo

    2015-01-01

    Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii. PMID:26544049

  10. Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

    SciTech Connect

    Gerthoffer, W.T.; Murphey, K.A.; Khoyi, M.A.

    1988-08-01

    Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased /sup 45/Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on /sup 45/Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.

  11. Contingent Involuntary Motoric Inhibition: The Involuntary Inhibition of a Motor Response Contingent on Top-Down Goals

    ERIC Educational Resources Information Center

    Anderson, Brian A.; Folk, Charles L.

    2012-01-01

    Effective motor control involves both the execution of appropriate responses and the inhibition of inappropriate responses that are evoked by response-associated stimuli. The inhibition of a motor response has traditionally been characterized as either a voluntary act of cognitive control or a low-level perceptual bias arising from processes such…

  12. Using voluntary motor commands to inhibit involuntary arm movements.

    PubMed

    Ghosh, Arko; Rothwell, John; Haggard, Patrick

    2014-11-01

    A hallmark of voluntary motor control is the ability to stop an ongoing movement. Is voluntary motor inhibition a general neural mechanism that can be focused on any movement, including involuntary movements, or is it mere termination of a positive voluntary motor command? The involuntary arm lift, or 'floating arm trick', is a distinctive long-lasting reflex of the deltoid muscle. We investigated how a voluntary motor network inhibits this form of involuntary motor control. Transcranial magnetic stimulation of the motor cortex during the floating arm trick produced a silent period in the reflexively contracting deltoid muscle, followed by a rebound of muscle activity. This pattern suggests a persistent generator of involuntary motor commands. Instructions to bring the arm down voluntarily reduced activity of deltoid muscle. When this voluntary effort was withdrawn, the involuntary arm lift resumed. Further, voluntary motor inhibition produced a strange illusion of physical resistance to bringing the arm down, as if ongoing involuntarily generated commands were located in a 'sensory blind-spot', inaccessible to conscious perception. Our results suggest that voluntary motor inhibition may be a specific neural function, distinct from absence of positive voluntary motor commands. PMID:25253453

  13. Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

    PubMed

    Kampourakis, Thomas; Yan, Ziqian; Gautel, Mathias; Sun, Yin-Biao; Irving, Malcolm

    2014-12-30

    Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

  14. Opaque1 Encodes a Myosin XI Motor Protein That Is Required for Endoplasmic Reticulum Motility and Protein Body Formation in Maize Endosperm[C][W][OA

    PubMed Central

    Wang, Guifeng; Wang, Fang; Wang, Gang; Wang, Fei; Zhang, Xiaowei; Zhong, Mingyu; Zhang, Jin; Lin, Dianbin; Tang, Yuanping; Xu, Zhengkai; Song, Rentao

    2012-01-01

    Myosins are encoded by multigene families and are involved in many basic biological processes. However, their functions in plants remain poorly understood. Here, we report the functional characterization of maize (Zea mays) opaque1 (o1), which encodes a myosin XI protein. o1 is a classic maize seed mutant with an opaque endosperm phenotype but a normal zein protein content. Compared with the wild type, o1 endosperm cells display dilated endoplasmic reticulum (ER) structures and an increased number of smaller, misshapen protein bodies. The O1 gene was isolated by map-based cloning and was shown to encode a member of the plant myosin XI family (myosin XI-I). In endosperm cells, the O1 protein is associated with rough ER and protein bodies. Overexpression of the O1 tail domain (the C-terminal 644 amino acids) significantly inhibited ER streaming in tobacco (Nicotiana benthamiana) cells. Yeast two-hybrid analysis suggested an association between O1 and the ER through a heat shock protein 70–interacting protein. In summary, this study indicated that O1 influences protein body biogenesis by affecting ER morphology and motility, ultimately affecting endosperm texture. PMID:22892319

  15. 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

  16. Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V

    PubMed Central

    Rogers, Stephen L.; Karcher, Ryan L.; Roland, Joseph T.; Minin, Alexander A.; Steffen, Walter; Gelfand, Vladimir I.

    1999-01-01

    Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161–164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis. PMID:10491390

  17. Crystallographic findings on the internally uncoupled and near-rigor states of myosin: Further insights into the mechanics of the motor

    PubMed Central

    Himmel, D. M.; Gourinath , S.; Reshetnikova, L.; Shen, Y.; Szent-Györgyi, A. G.; Cohen, C.

    2002-01-01

    Here we report a 2.3-Å crystal structure of scallop myosin S1 complexed with ADP⋅BeFx, as well as three additional structures (at 2.8–3.8 Å resolution) for this S1 complexed with ATP analogs, some of which are cross-linked by para-phenyl dimaleimide, a short intramolecular cross-linker. In all cases, the complexes are characterized by an unwound SH1 helix first seen in an unusual 2.5-Å scallop myosin-MgADP structure and described as corresponding to a previously unrecognized actin-detached internally uncoupled state. The unwinding of the SH1 helix effectively uncouples the converter/lever arm module from the motor and allows cross-linking by para-phenyl dimaleimide, which has been shown to occur only in weak actin-binding states of the molecule. Mutations near the metastable SH1 helix that disable the motor can be accounted for by viewing this structural element as a clutch controlling the transmission of torque to the lever arm. We have also determined a 3.2-Å nucleotide-free structure of scallop myosin S1, which suggests that in the near-rigor state there are two conformations in the switch I loop, depending on whether nucleotide is present. Analysis of the subdomain motions in the weak actin-binding states revealed by x-ray crystallography, together with recent electron microscopic results, clarify the mechanical roles of the parts of the motor in the course of the contractile cycle and suggest how strong binding to actin triggers both the power stroke and product release. PMID:12297624

  18. She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p

    PubMed Central

    Böhl, Florian; Kruse, Claudia; Frank, Andrea; Ferring, Dunja; Jansen, Ralf-Peter

    2000-01-01

    RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences (‘zipcodes’) within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p’s C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA–motor protein adapters. PMID:11032818

  19. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

    PubMed

    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  20. Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

    PubMed Central

    Bi, Guo-Qiang; Morris, Robert L.; Liao, Guochun; Alderton, Janet M.; Scholey, Jonathan M.; Steinhardt, Richard A.

    1997-01-01

    Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca2+-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca2+/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca2+-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain. PMID:9281579

  1. Transport of ER vesicles on actin filaments in neurons by myosin V.

    PubMed

    Tabb, J S; Molyneaux, B J; Cohen, D L; Kuznetsov, S A; Langford, G M

    1998-11-01

    Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for

  2. Are Motor Skills and Motor Inhibitions Impaired in Tourette Syndrome? A Review

    PubMed Central

    Kalsi, Navkiran; Tambelli, Renata; Aceto, Paola; Lai, Carlo

    2015-01-01

    Tourette syndrome (TS) is a neurodevelopmental motor disorder described as an inability to inhibit unwanted motor movements. This article reviews research on the execution and inhibition of voluntary motor movements in TS. Over last two decades, a number of studies have addressed the structural and functional deficits associated with this syndrome. Only a limited number of studies have assessed the motor skills in these patients but have failed to reach any conclusive outcome. In the domain of response inhibition also, studies have reported arguable impairments in these patients. It is suggested that these conflicting results can be attributed to co-occurring comorbid conditions, the constraints posed by variable age groups, lack of control measures, and lack of specificity of domains addressed. This review will describe a way in which future research can be directed to increase our knowledge of this otherwise complex spectrum of disorders. PMID:26279630

  3. 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.

  4. 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

  5. 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. PMID:27597808

  6. 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.

  7. Detecting age differences in inhibition processes with a test of perceptual and motor inhibition

    PubMed Central

    Jennings, J. Richard; Mendelson, David N.; Redfern, Mark S.; Nebes, Robert D

    2010-01-01

    We asked whether different forms of inhibition are altered differently by aging using a Motor and Perceptual Inhibition Test (MAPIT) based on Nassauer and Halperin (Nassauer & Halperin, 2003). Ninety-eight individuals participating in studies of balance and attention were separated into younger (mean age 25 years) and older participants (mean age 73). Older participants showed less Perceptual and Motor Inhibition than younger participant with moderation of this effect by gender. The two scores were uncorrelated in the young but significantly correlated in the older group. Overall, the MAPIT appeared to yield reliable measures of two aspects of inhibition that demonstrate a differential impact of age. PMID:21424956

  8. 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. PMID:27548633

  9. Alpha oscillatory correlates of motor inhibition in the aged brain

    PubMed Central

    Bönstrup, Marlene; Hagemann, Julian; Gerloff, Christian; Sauseng, Paul; Hummel, Friedhelm C.

    2015-01-01

    Exerting inhibitory control is a cognitive ability mediated by functions known to decline with age. The goal of this study is to add to the mechanistic understanding of cortical inhibition during motor control in aged brains. Based on behavioral findings of impaired inhibitory control with age we hypothesized that elderly will show a reduced or a lack of EEG alpha-power increase during tasks that require motor inhibition. Since inhibitory control over movements has been shown to rely on prior motor memory formation, we investigated cortical inhibitory processes at two points in time—early after learning and after an overnight consolidation phase and hypothesized an overnight increase of inhibitory capacities. Young and elderly participants acquired a complex finger movement sequence and in each experimental session brain activity during execution and inhibition of the sequence was recorded with multi-channel EEG. We assessed cortical processes of sustained inhibition by means of task-induced changes of alpha oscillatory power. During inhibition of the learned movement, young participants showed a significant alpha power increase at the sensorimotor cortices whereas elderly did not. Interestingly, for both groups, the overnight consolidation phase improved up-regulation of alpha power during sustained inhibition. This points to deficits in the generation and enhancement of local inhibitory mechanisms at the sensorimotor cortices in aged brains. However, the alpha power increase in both groups implies neuroplastic changes that strengthen the network of alpha power generation over time in young as well as elderly brains. PMID:26528179

  10. 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.

  11. Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins.

    PubMed

    Seki, Masaya; Kashiyama, Taku; Hachikubo, You; Ito, Kohji; Yamamoto, Keiichi

    2004-11-19

    A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.

  12. Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

    PubMed Central

    Root, D D; Reisler, E

    1992-01-01

    The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin. PMID:1420910

  13. Unique sequences and predicted functions of myosins in Tetrahymena thermophila.

    PubMed

    Sugita, Maki; Iwataki, Yoshinori; Nakano, Kentaro; Numata, Osamu

    2011-07-01

    Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.

  14. Directional Mechanosensing in Myosin VI

    NASA Astrophysics Data System (ADS)

    Yang, Yubo; Tehver, Riina

    2013-03-01

    Myosin is a family of versatile motor proteins that perform various tasks, such as organelle transport, anchoring and cell deformation. Although the general mechanism of the motors has been fairly well established, details on dynamic aspects like force response of the motor, and force propagation are yet to be fully understood. In this poster, we present the response of the ATP binding region to force exerted on the tail domain in order to test the proposed tension-dependent gating mechanism of myosin VI processive motion. We employed the Self-Organized Polymer model in a computer simulation to explore the effect. Current results show that the ATP binding domain of myosin VI indeed exhibits tension dependence - both structurally and dynamically.

  15. Thiol reactivity as a sensor of rotation of the converter in myosin.

    PubMed

    Onishi, Hirofumi; Nitanai, Yasushi

    2008-04-25

    Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the "converter", which rotates by approximately 70 degrees upon the transition from the "nucleotide-free" state to the "pre-power stroke" state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, "SH1", was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.

  16. The myosin inhibitor blebbistatin stabilizes the super-relaxed state in skeletal muscle.

    PubMed

    Wilson, Clyde; Naber, Nariman; Pate, Edward; Cooke, Roger

    2014-10-01

    The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX. PMID:25296316

  17. Evaluation of Acanthamoeba myosin-IC as a potential therapeutic target.

    PubMed

    Martín-Navarro, Carmen M; Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E; Valladares, Basilio; Maciver, Sutherland K

    2014-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as "traction-mediated cytokinesis". We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

  18. Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

    PubMed Central

    Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E.; Valladares, Basilio; Maciver, Sutherland K.

    2014-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as “traction-mediated cytokinesis”. We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

  19. Molecular engineering of myosin.

    PubMed Central

    Manstein, Dietmar J

    2004-01-01

    Protein engineering and design provide excellent tools to investigate the principles by which particular structural features relate to the mechanisms that underlie the biological function of a protein. In addition to studies aimed at dissecting the communication pathways within enzymes, recent advances in protein engineering approaches make it possible to generate enzymes with increased catalytic efficiency and specifically altered or newly introduced functions. Here, two approaches using state-of-the-art protein design and engineering are described in detail to demonstrate how key features of the myosin motor can be changed in a specific and predictable manner. First, it is shown how replacement of an actin-binding surface loop with synthetic sequences, whose flexibility and charge density is varied, can be employed to manipulate the actin affinity, the catalytic activity and the efficiency of coupling between actin- and nucleotide-binding sites of myosin motor constructs. Then the use of pre-existing molecular building blocks, which are derived from unrelated proteins, is described for manipulating the velocity and even the direction of movement of recombinant myosins. PMID:15647166

  20. Myosins in protists.

    PubMed

    Gavin, R H

    2001-01-01

    This review focuses on selected papers that illustrate an historical perspective and the current knowledge of myosin structure and function in protists. The review contains a general description of myosin structure, a phylogenetic tree of the myosin classes, and descriptions of myosin isoforms identified in protists. Each myosin is discussed within the context of the taxonomic group of the organism in which the myosin has been identified. Domain structure, cellular location, function, and regulation are described for each myosin.

  1. A Role for Myosin V Motor Proteins in the Selective Delivery of Kv Channel Isoforms to the Membrane Surface of Cardiac Myocytes

    PubMed Central

    Schumacher-Bass, Sarah M.; Vesely, Eileen D.; Zhang, Lian; Ryland, Katherine E.; McEwen, Dyke P.; Chan, Priscilla J.; Frasier, Chad R.; McIntyre, Jeremy C.; Shaw, Robin M.; Martens, Jeffrey R.

    2014-01-01

    Rationale Kv1.5 (KCNA5) mediates the IKur current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation (AF), Kv1.5 has emerged as a promising target for the treatment of AF. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. Objective To investigate the role of the unconventional myosin V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. Methods and Results Western Blot analysis showed MYO5A and MYO5B expression in the heart, while disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and shRNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and Connexin-43 (Cx43), but not hERG (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, while MYO5B regulated post-endocytic recycling. Over-expression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. Conclusions MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current, and consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias. PMID:24508725

  2. Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

    PubMed

    Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

    2015-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that

  3. All-Trans Retinoic Acid Inhibits Human Colorectal Cancer Cells RKO Migration via Downregulating Myosin Light Chain Kinase Expression through MAPK Signaling Pathway.

    PubMed

    Zuo, Li; Yang, Xiaoping; Lu, Man; Hu, Ruolei; Zhu, Huaqing; Zhang, Sumei; Zhou, Qing; Chen, Feihu; Gui, Shuyu; Wang, Yuan

    2016-10-01

    All-trans-retinoic acid (ATRA) inhibits the invasive and metastatic potentials of various cancer cells. However, the underlying mechanism is unclear. Here, we demonstrate that ATRA inhibited colorectal cancer cells RKO (human colon adenocarcinoma cell) migration by downregulating cell movement and increasing cell adhesion. ATRA inhibited the expression and activation of myosin light chain kinase (MLCK) in RKO cells, while the expression level of MLC phosphatase (MLCP) had no change in RKO cells treated with or without ATRA. The expression and activity of MLC was also inhibited in RKO cells exposed to ATRA. Intriguingly, ATRA increased the expression of occludin messenger RNA (mRNA) and protein and its localization on cell membrane. However, ATRA did not change the expression of zonula occludens 1 (ZO-1), but increased the accumulation of ZO-1 on RKO cells membrane. ML-7, an inhibitor of MLCK, significantly inhibited RKO cell migration. Furthermore, knockdown of endogenous MLCK expression inhibited RKO migration. Mechanistically, we showed that MAPK-specific inhibitor PD98059 enhanced the inhibitory effect of ATRA on RKO migration. In contrast, phorbol 12-myristate 13-acetate (PMA) attenuated the effects of ATRA in RKO cells. Moreover, knocking down endogenous extracellular signal-regulated kinase (ERK) expression inhibited MLCK expression in the RKO cells. In conclusion, ATRA inhibits RKO migration by reducing MLCK expression via extracellular signal-regulated kinase 1/Mitogen-activated protein kinase (ERK1/MAPK) signaling pathway. PMID:27564600

  4. A mechanochemical model for myosin VI

    NASA Astrophysics Data System (ADS)

    Tehver, Riina; Jack, Amanda; Lowe, Ian

    Myosin VI is a motor protein that transports cellular cargo along actin filaments. This transport takes place as a result of a coordinated mechano-chemical cycle that is controlled by external variables including imposed force and nucleotide concentrations. We present a model that captures the different dynamic pathways that myosin VI can take in response to these variables. The results of our model for experimentally observable quantities, such as the motor velocity or run length, agree with available experimental data, and we can also make predictions beyond the tested regimes. Using the model, we study how myosin VI reacts to its environment and test its operational efficiency.

  5. The myosin motor domain-containing chitin synthase PdChsVII is required for development, cell wall integrity and virulence in the citrus postharvest pathogen Penicillium digitatum.

    PubMed

    Gandía, Mónica; Harries, Eleonora; Marcos, Jose F

    2014-06-01

    Chitin is an essential component of the fungal cell wall and a potential target in the development of new antifungal compounds, due to its presence in fungi and not in plants or vertebrates. Chitin synthase genes (chs) constitute a complex family in filamentous fungi and are involved in fungal development, morphogenesis, pathogenesis and virulence. In this study, additional chs genes in the citrus postharvest pathogen Penicillium digitatum have been identified. Comparative analyses included each PdChs in each one of the classes I to VII previously established, and support the grouping of these into three divisions. Disruption of the gene coding PdChsVII, which contains a short version of a myosin motor domain, has been achieved by using Agrobacterium tumefaciens-mediated transformation and revealed its role in the life cycle of the fungus. Disruption strains were viable but showed reduced growth and conidia production. Moreover, Pdchs mutants developed morphological defects as balloon-like enlarged cells and increased chitin content, indicative of an altered cell wall structure. Gene disruption also increased susceptibility to antifungal compounds such as calcofluor white (CFW), sodium dodecyl sulfate (SDS), hydroxide peroxide (H2O2) and commercial fungicides, but significantly no change was observed in the sensitivity to antifungal peptides. The PdchsVII mutants were able to infect citrus fruit and produced tissue maceration, although had reduced virulence and most importantly were greatly impaired in the production of visible mycelium and conidia on the fruit.

  6. Kinetic and Motor Functions Mediated by Distinct Regions of the Regulatory Light Chain of Smooth Muscle Myosin1,2

    PubMed Central

    Ni, Shaowei; Hong, Feng; Brewer, Paul D.; Ikebe, Mitsuo; Onishi, Hirofumi; Baker, Jonathan E.; Facemyer, Kevin C.; Cremo, Christine R.

    2009-01-01

    To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP95-98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg2+-ATPase (Vmax and Katpase) and in vitro actin sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the upstate (0.007-0.030 s-1) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (Katpase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the Vmax was 64% and actin sliding velocity was ∼50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation. PMID:19635597

  7. Myosin II Dynamics during Embryo Morphogenesis

    NASA Astrophysics Data System (ADS)

    Kasza, Karen

    2013-03-01

    During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

  8. New insights into myosin evolution and classification.

    PubMed

    Foth, Bernardo J; Goedecke, Marc C; Soldati, Dominique

    2006-03-01

    Myosins are eukaryotic actin-dependent molecular motors important for a broad range of functions like muscle contraction, vision, hearing, cell motility, and host cell invasion of apicomplexan parasites. Myosin heavy chains consist of distinct head, neck, and tail domains and have previously been categorized into 18 different classes based on phylogenetic analysis of their conserved heads. Here we describe a comprehensive phylogenetic examination of many previously unclassified myosins, with particular emphasis on sequences from apicomplexan and other chromalveolate protists including the model organism Toxoplasma, the malaria parasite Plasmodium, and the ciliate Tetrahymena. Using different phylogenetic inference methods and taking protein domain architectures, specific amino acid polymorphisms, and organismal distribution into account, we demonstrate a hitherto unrecognized common origin for ciliate and apicomplexan class XIV myosins. Our data also suggest common origins for some apicomplexan myosins and class VI, for classes II and XVIII, for classes XII and XV, and for some microsporidian myosins and class V, thereby reconciling evolutionary history and myosin structure in several cases and corroborating the common coevolution of myosin head, neck, and tail domains. Six novel myosin classes are established to accommodate sequences from chordate metazoans (class XIX), insects (class XX), kinetoplastids (class XXI), and apicomplexans and diatom algae (classes XXII, XXIII, and XXIV). These myosin (sub)classes include sequences with protein domains (FYVE, WW, UBA, ATS1-like, and WD40) previously unknown to be associated with myosin motors. Regarding the apicomplexan "myosome," we significantly update class XIV classification, propose a systematic naming convention, and discuss possible functions in these parasites.

  9. Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis.

    PubMed

    Iwasaki, Takahiro; Katayama, Takeshi; Kohama, Kazuhiro; Endo, Yaeta; Sawasaki, Tatsuya

    2013-03-01

    In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

  10. Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

    PubMed Central

    Wong, Raymond; Fabian, Lacramioara; Forer, Arthur; Brill, Julie A

    2007-01-01

    Background Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring. PMID:17509155

  11. A Model of Motor Inhibition for a Complex Skill: Baseball Batting

    ERIC Educational Resources Information Center

    Gray, Rob

    2009-01-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different…

  12. GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

    PubMed

    Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

    2011-09-22

    The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons.

  13. Brief report: Response inhibition and processing speed in children with motor difficulties and developmental coordination disorder.

    PubMed

    Bernardi, Marialivia; Leonard, Hayley C; Hill, Elisabeth L; Henry, Lucy A

    2016-01-01

    A previous study reported that children with poor motor skills, classified as having motor difficulties (MD) or Developmental Coordination Disorder (DCD), produced more errors in a motor response inhibition task compared to typically developing (TD) children but did not differ in verbal inhibition errors. The present study investigated whether these groups differed in the length of time they took to respond in order to achieve these levels of accuracy, and whether any differences in response speed could be explained by generally slow information processing in children with poor motor skills. Timing data from the Verbal Inhibition Motor Inhibition test were analyzed to identify differences in performance between the groups on verbal and motor inhibition, as well as on processing speed measures from standardized batteries. Although children with MD and DCD produced more errors in the motor inhibition task than TD children, the current analyses found that they did not take longer to complete the task. Children with DCD were slower at inhibiting verbal responses than TD children, while the MD group seemed to perform at an intermediate level between the other groups in terms of verbal inhibition speed. Slow processing speed did not account for these group differences. Results extended previous research into response inhibition in children with poor motor skills by explicitly comparing motor and verbal responses, and suggesting that slow performance, even when accurate, may be attributable to an inefficient way of inhibiting responses, rather than slow information processing speed per se.

  14. All-atom molecular dynamics simulations of actin-myosin interactions: a comparative study of cardiac α myosin, β myosin, and fast skeletal muscle myosin.

    PubMed

    Li, Minghui; Zheng, Wenjun

    2013-11-26

    Myosins are a superfamily of actin-binding motor proteins with significant variations in kinetic properties (such as actin binding affinity) between different isoforms. It remains unknown how such kinetic variations arise from the structural and dynamic tuning of the actin-myosin interface at the amino acid residue level. To address this key issue, we have employed molecular modeling and simulations to investigate, with atomistic details, the isoform dependence of actin-myosin interactions in the rigor state. By combining electron microscopy-based docking with homology modeling, we have constructed three all-atom models for human cardiac α and β and rabbit fast skeletal muscle myosin in complex with three actin subunits in the rigor state. Starting from these models, we have performed extensive all-atom molecular dynamics (MD) simulations (total of 100 ns per system) and then used the MD trajectories to calculate actin-myosin binding free energies with contributions from both electrostatic and nonpolar forces. Our binding calculations are in good agreement with the experimental finding of isoform-dependent differences in actin binding affinity between these myosin isoforms. Such differences are traced to changes in actin-myosin electrostatic interactions (i.e., hydrogen bonds and salt bridges) that are highly dynamic and involve several flexible actin-binding loops. By partitioning the actin-myosin binding free energy to individual myosin residues, we have also identified key myosin residues involved in the actin-myosin interactions, some of which were previously validated experimentally or implicated in cardiomyopathy mutations, and the rest make promising targets for future mutational experiments. PMID:24224850

  15. Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries

    PubMed Central

    Moreno-Domínguez, Alejandro; Colinas, Olaia; El-Yazbi, Ahmed; Walsh, Emma J; Hill, Michael A; Walsh, Michael P; Cole, William C

    2013-01-01

    The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca2+ entry via voltage-gated Ca2+ channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca2+ sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate

  16. Ca2+ sensitization due to myosin light chain phosphatase inhibition and cytoskeletal reorganization in the myogenic response of skeletal muscle resistance arteries.

    PubMed

    Moreno-Domínguez, Alejandro; Colinas, Olaia; El-Yazbi, Ahmed; Walsh, Emma J; Hill, Michael A; Walsh, Michael P; Cole, William C

    2013-03-01

    Abstract  The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca(2+) entry via voltage-gated Ca(2+) channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca(2+) sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca(2+) sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our

  17. Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

    PubMed Central

    Reddy, Anireddy SN; Day, Irene 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. PMID:11516337

  18. Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles*

    PubMed Central

    Nagy, Stanislav; Rock, Ronald S.

    2010-01-01

    Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, we employed a domain-swapping approach with the nonselective myosin V to identify the selectivity module of myosin X. We found a surprising role of the myosin X tail region (post-IQ) in supporting long runs on bundles. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses. PMID:20538587

  19. Imagining is Not Doing but Involves Specific Motor Commands: A Review of Experimental Data Related to Motor Inhibition

    PubMed Central

    Guillot, Aymeric; Di Rienzo, Franck; MacIntyre, Tadhg; Moran, Aidan; Collet, Christian

    2012-01-01

    There is now compelling evidence that motor imagery (MI) and actual movement share common neural substrate. However, the question of how MI inhibits the transmission of motor commands into the efferent pathways in order to prevent any movement is largely unresolved. Similarly, little is known about the nature of the electromyographic activity that is apparent during MI. In addressing these gaps in the literature, the present paper argues that MI includes motor execution commands for muscle contractions which are blocked at some level of the motor system by inhibitory mechanisms. We first assemble data from neuroimaging studies that demonstrate that the neural networks mediating MI and motor performance are not totally overlapping, thereby highlighting potential differences between MI and actual motor execution. We then review MI data indicating the presence of subliminal muscular activity reflecting the intrinsic characteristics of the motor command as well as increased corticomotor excitability. The third section not only considers the inhibitory mechanisms involved during MI but also examines how the brain resolves the problem of issuing the motor command for action while supervising motor inhibition when people engage in voluntary movement during MI. The last part of the paper draws on imagery research in clinical contexts to suggest that some patients move while imagining an action, although they are not aware of such movements. In particular, experimental data from amputees as well as from patients with Parkinson’s disease are discussed. We also review recent studies based on comparing brain activity in tetraplegic patients with that from healthy matched controls that provide insights into inhibitory processes during MI. We conclude by arguing that based on available evidence, a multifactorial explanation of motor inhibition during MI is warranted. PMID:22973214

  20. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

    PubMed

    van der Honing, Hannie S; de Ruijter, Norbert C A; Emons, Anne Mie C; Ketelaar, Tijs

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

  1. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  2. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    PubMed

    Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  3. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development

    PubMed Central

    Peremyslov, Valera V.; Cole, Rex A.; Fowler, John E.; Dolja, Valerian V.

    2015-01-01

    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  4. Cognitive Control Reflects Context Monitoring, Not Motoric Stopping, in Response Inhibition

    PubMed Central

    Chatham, Christopher H.; Claus, Eric D.; Kim, Albert; Curran, Tim; Banich, Marie T.; Munakata, Yuko

    2012-01-01

    The inhibition of unwanted behaviors is considered an effortful and controlled ability. However, inhibition also requires the detection of contexts indicating that old behaviors may be inappropriate – in other words, inhibition requires the ability to monitor context in the service of goals, which we refer to as context-monitoring. Using behavioral, neuroimaging, electrophysiological and computational approaches, we tested whether motoric stopping per se is the cognitively-controlled process supporting response inhibition, or whether context-monitoring may fill this role. Our results demonstrate that inhibition does not require control mechanisms beyond those involved in context-monitoring, and that such control mechanisms are the same regardless of stopping demands. These results challenge dominant accounts of inhibitory control, which posit that motoric stopping is the cognitively-controlled process of response inhibition, and clarify emerging debates on the frontal substrates of response inhibition by replacing the centrality of controlled mechanisms for motoric stopping with context-monitoring. PMID:22384038

  5. Deficient grip force control in schizophrenia: behavioral and modeling evidence for altered motor inhibition and motor noise.

    PubMed

    Teremetz, Maxime; Amado, Isabelle; Bendjemaa, Narjes; Krebs, Marie-Odile; Lindberg, Pavel G; Maier, Marc A

    2014-01-01

    Whether upper limb sensorimotor control is affected in schizophrenia and how underlying pathological mechanisms may potentially intervene in these deficits is still being debated. We tested voluntary force control in schizophrenia patients and used a computational model in order to elucidate potential cerebral mechanisms underlying sensorimotor deficits in schizophrenia. A visuomotor grip force-tracking task was performed by 17 medicated and 6 non-medicated patients with schizophrenia (DSM-IV) and by 15 healthy controls. Target forces in the ramp-hold-and-release paradigm were set to 5 N and to 10% maximal voluntary grip force. Force trajectory was analyzed by performance measures and Principal Component Analysis (PCA). A computational model incorporating neural control signals was used to replicate the empirically observed motor behavior and to explore underlying neural mechanisms. Grip task performance was significantly lower in medicated and non-medicated schizophrenia patients compared to controls. Three behavioral variables were significantly higher in both patient groups: tracking error (by 50%), coefficient of variation of force (by 57%) and duration of force release (up by 37%). Behavioral performance did not differ between patient groups. Computational simulation successfully replicated these findings and predicted that decreased motor inhibition, together with an increased signal-dependent motor noise, are sufficient to explain the observed motor deficits in patients. PCA also suggested altered motor inhibition as a key factor differentiating patients from control subjects: the principal component representing inhibition correlated with clinical severity. These findings show that schizophrenia affects voluntary sensorimotor control of the hand independent of medication, and suggest that reduced motor inhibition and increased signal-dependent motor noise likely reflect key pathological mechanisms of the sensorimotor deficit.

  6. Association of a Nonmuscle Myosin II with Axoplasmic Organelles

    PubMed Central

    DeGiorgis, Joseph A.; Reese, Thomas S.; Bearer, Elaine L.

    2002-01-01

    Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor. PMID:11907281

  7. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length.

    PubMed

    Schott, Daniel H; Collins, Ruth N; Bretscher, Anthony

    2002-01-01

    Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.

  8. CORRELATIONS OF PESTICIDE-INDUCED CHOLINESTERASE INHIBITION AND MOTOR ACTIVITY CHANGES IN ADULT RATS.

    EPA Science Inventory

    The acute neurobehavioral effects of acetylcholinesterase-inhibiting pesticides are primarily due to overstimulation of the cholinergic system. Lowered motor activity levels represent a sensitive endpoint with which to monitor functional changes in laboratory animals exposed to ...

  9. Lipid Emulsion Inhibits Vasodilation Induced by a Toxic Dose of Bupivacaine via Attenuated Dephosphorylation of Myosin Phosphatase Target Subunit 1 in Isolated Rat Aorta.

    PubMed

    Ok, Seong-Ho; Byon, Hyo-Jin; Kwon, Seong-Chun; Park, Jungchul; Lee, Youngju; Hwang, Yeran; Baik, Jiseok; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2015-01-01

    Lipid emulsions are widely used for the treatment of systemic toxicity that arises from local anesthetics. The goal of this in vitro study was to examine the cellular mechanism associated with the lipid emulsion-mediated attenuation of vasodilation induced by a toxic dose of bupivacaine in isolated endothelium-denuded rat aorta. The effects of lipid emulsion on vasodilation induced by bupivacaine, mepivacaine, and verapamil were assessed in isolated aorta precontracted with phenylephrine, the Rho kinase stimulant NaF, and the protein kinase C activator phorbol 12,13-dibutyrate (PDBu). The effects of Rho kinase inhibitor Y-27632 on contraction induced by phenylephrine or NaF were assessed. The effects of bupivacaine on intracellular calcium concentrations ([Ca(2+)]i) and tension induced by NaF were simultaneously measured. The effects of bupivacaine alone and lipid emulsion plus bupivacaine on myosin phosphatase target subunit 1 (MYPT1) phosphorylation induced by NaF were examined in rat aortic vascular smooth muscle cells. In precontracted aorta, the lipid emulsion attenuated bupivacaine-induced vasodilation but had no effect on mepivacaine-induced vasodilation. Y-27632 attenuated contraction induced by either phenylephrine or NaF. The lipid emulsion attenuated verapamil-induced vasodilation. Compared with phenylephrine-induced precontracted aorta, bupivacaine-induced vasodilation was slightly attenuated in NaF-induced precontracted aorta. The magnitude of the bupivacaine-induced vasodilation was higher than that of a bupivacaine-induced decrease in [Ca(2+)]i. Bupivacaine attenuated NaF-induced MYPT1 phosphorylation, whereas lipid emulsion pretreatment attenuated the bupivacaine-induced inhibition of MYPT1 phosphorylation induced by NaF. Taken together, these results suggest that lipid emulsions attenuate bupivacaine-induced vasodilation via the attenuation of inhibition of MYPT1 phosphorylation evoked by NaF.

  10. Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractility.

    PubMed

    Aifuwa, Ivie; Giri, Anjil; Longe, Nick; Lee, Sang Hyuk; An, Steven S; Wirtz, Denis

    2015-10-13

    Cells induced into senescence exhibit a marked increase in the secretion of pro-inflammatory cytokines termed senescence-associated secretory phenotype (SASP). Here we report that SASP from senescent stromal fibroblasts promote spontaneous morphological changes accompanied by an aggressive migratory behavior in originally non-motile human breast cancer cells. This phenotypic switch is coordinated, in space and time, by a dramatic reorganization of the actin and microtubule filament networks, a discrete polarization of EB1 comets, and an unconventional front-to-back inversion of nucleus-MTOC polarity. SASP-induced morphological/migratory changes are critically dependent on microtubule integrity and dynamics, and are coordinated by the inhibition of RhoA and cell contractility. RhoA/ROCK inhibition reduces focal adhesions and traction forces, while promoting a novel gliding mode of migration. PMID:26483365

  11. Myosin flexibility: structural domains and collective vibrations.

    PubMed

    Navizet, Isabelle; Lavery, Richard; Jernigan, Robert L

    2004-02-15

    The movement of the myosin motor along an actin filament involves a directed conformational change within the cross-bridge formed between the protein and the filament. Despite the structural data that has been obtained on this system, little is known of the mechanics of this conformational change. We have used existing crystallographic structures of three conformations of the myosin head, containing the motor domain and the lever arm, for structural comparisons and mechanical studies with a coarse-grained elastic network model. The results enable us to define structurally conserved domains within the protein and to better understand myosin flexibility. Notably they point to the role of the light chains in rigidifying the lever arm and to changes in flexibility as a consequence of nucleotide binding.

  12. Myosins and cell dynamics in cellular slime molds.

    PubMed

    Yumura, Shigehiko; Uyeda, Taro Q P

    2003-01-01

    Myosin is a mechanochemical transducer and serves as a motor for various motile activities such as cell migration, cytokinesis, maintenance of cell shape, phagocytosis, and morphogenesis. Nonmuscle myosin in vivo does not either stay static at specific subcellular regions or construct highly organized structures, such as sarcomere in skeletal muscle cells. The cellular slime mold Dictyostelium discoideum is an ideal "model organism" for the investigation of cell movement and cytokinesis. The advantages of this organism prompted researchers to carry out pioneering cell biological, biochemical, and molecular genetic studies on myosin II, which resulted in elucidation of many fundamental features of function and regulation of this most abundant molecular motor. Furthermore, recent molecular biological research has revealed that many unconventional myosins play various functions in vivo. In this article, how myosins are organized and regulated in a dynamic manner in Dictyostelium cells is reviewed and discussed. PMID:12722951

  13. 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

  14. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  15. Myosin-V stepping kinetics: a molecular model for processivity.

    PubMed

    Rief, M; Rock, R S; Mehta, A D; Mooseker, M S; Cheney, R E; Spudich, J A

    2000-08-15

    Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

  16. EPR and fluorescence depolarization studies on bovine cardiac myosin.

    PubMed

    Stone, D B; Mendelson, R A; Botts, J; Cheung, P H

    1981-09-01

    To test for possible differences in local conformation and S1 flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS) and a spin label having iodoacetamide reactivity. The marked activation of the Ca2+-ATPase (6- to 8-fold) and inhibition of the K+ (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH1) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in steric restraints around the SH1 thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M.ADP and M.ADP.Pi. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kd (ADP) = 7 microM, n = 1.6. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins.

  17. Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

    PubMed Central

    Kaiser, Christian M.; Bujalowski, Paul J.; Ma, Liang; Anderson, John; Epstein, Henry F.; Oberhauser, Andres F.

    2012-01-01

    Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail. PMID:22824286

  18. Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

    ERIC Educational Resources Information Center

    Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

    2004-01-01

    To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

  19. [C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin].

    PubMed

    Pronina, O E; Copeland, O; Marston, S; Borovikov, Iu S

    2006-01-01

    Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.

  20. 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.

  1. 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. PMID:26116789

  2. Four things to know about myosin light chains as reporters for non-muscle myosin-2 dynamics in live cells.

    PubMed

    Heissler, Sarah M; Sellers, James R

    2015-02-01

    The interplay between non-muscle myosins-2 and filamentous actin results in cytoplasmic contractility which is essential for eukaryotic life. Concomitantly, there is tremendous interest in elucidating the physiological function and temporal localization of non-muscle myosin-2 in cells. A commonly used method to study the function and localization of non-muscle myosin-2 is to overexpress a fluorescent protein (FP)-tagged version of the regulatory light chain (RLC) which binds to the myosin-2 heavy chain by mass action. Caveats about this approach include findings from recent studies indicating that the RLC does not bind exclusively to the non-muscle myosin-2 heavy chain. Rather, it can also associate with the myosin heavy chains of several other classes as well as other targets than myosin. In addition, the presence of the FP moiety may compromise myosin's enzymatic and mechanical performance. This and other factors to be discussed in this commentary raise questions about the possible complications in using FP-RLC as a marker for the dynamic localization and regulatory aspects of non-muscle myosin-2 motor functions in cell biological experiments.

  3. A model of motor inhibition for a complex skill: baseball batting.

    PubMed

    Gray, Rob

    2009-06-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different inhibition signals (out of range launch angle, early expected-actual trajectory discrepancy, and late expected-actual trajectory discrepancy) resulting in four possible response outcomes for the batter's swing (full swing, inhibited swing, partial response, or interrupted swing). The predictions of the model were compared with the actual batting performance of 20 baseball players using a high-fidelity batting simulator. The proportions of the different response outcomes could be explained by the inhibition model for 17/20 of the batters in the study. These findings suggest that models of motor inhibition developed for simple, discrete tasks can be applied to complex, multistage behaviors. This batting inhibition model could be used to provide a quantitative measure of a player's bat control for training and player-screening purposes. (PsycINFO Database Record (c) 2009 APA, all rights reserved). PMID:19586249

  4. A model of motor inhibition for a complex skill: baseball batting.

    PubMed

    Gray, Rob

    2009-06-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different inhibition signals (out of range launch angle, early expected-actual trajectory discrepancy, and late expected-actual trajectory discrepancy) resulting in four possible response outcomes for the batter's swing (full swing, inhibited swing, partial response, or interrupted swing). The predictions of the model were compared with the actual batting performance of 20 baseball players using a high-fidelity batting simulator. The proportions of the different response outcomes could be explained by the inhibition model for 17/20 of the batters in the study. These findings suggest that models of motor inhibition developed for simple, discrete tasks can be applied to complex, multistage behaviors. This batting inhibition model could be used to provide a quantitative measure of a player's bat control for training and player-screening purposes. (PsycINFO Database Record (c) 2009 APA, all rights reserved).

  5. Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

    PubMed Central

    Stauffer, Eric A.; Scarborough, John D.; Hirono, Moritoshi; Miller, Emilie D.; Shah, Kavita; Mercer, John A.; Holt, Jeffrey R.; Gillespie, Peter G.

    2009-01-01

    Summary In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells. PMID:16102537

  6. Structural insight into the UNC-45-myosin complex.

    PubMed

    Fratev, Filip; Osk Jónsdóttir, Svava; Pajeva, Ilza

    2013-07-01

    The UNC-45 chaperone protein interacts with and affects the folding, stability, and the ATPase activity of myosins. It plays a critical role in the cardiomyopathy development and in the breast cancer tumor growth. Here we propose the first structural model of the UNC-45-myosin complex using various in silico methods. Initially, the human UNC-45B binding epitope was identified and the protein was docked to the cardiac myosin (MYH7) motor domain. The final UNC45B-MYH7 structure was obtained by performing of total 630 ns molecular dynamics simulations. The results indicate a complex formation, which is mainly stabilized by electrostatic interactions. Remarkably, the contact surface area is similar to that of the myosin-actin complex. A significant interspecies difference in the myosin binding epitope is observed. Our results reveal the structural basis of MYH7 exons 15-16 hypertrophic cardiomyopathy mutations and provide directions for drug targeting. PMID:23408646

  7. Renshaw cells are inactive during motor inhibition elicited by the pontine microinjection of carbachol.

    PubMed

    Morales, F R; Engelhardt, J K; Pereda, A E; Yamuy, J; Chase, M H

    1988-04-12

    The present study was undertaken to determine whether the postsynaptic inhibition of motoneurons that occurs following the pontine microinjection of carbachol in the decerebrate cat is due to the activity of Renshaw cells. Thirty-two out of 37 Renshaw cells (86%) were spontaneously active prior to the administration of carbachol, whereas only 2 out of 13 Renshaw cells (15%) discharged during carbachol-induced motor inhibition. In addition, discrete inhibitory synaptic potentials were observed in 33% of the Renshaw cells from which intracellular recordings were obtained after carbachol administration, indicating that these cells were actively inhibited. The finding that a population of Renshaw cells, which inhibit motoneurons, were themselves inhibited during a period of profound motoneuron inhibition was quite unexpected. These results support the conclusion that Renshaw cells are not the inhibitory interneurons that are responsible for the powerful inhibition of motoneurons that occurs following the pontine microinjection of carbachol. PMID:3380320

  8. Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

    PubMed Central

    Johnson, Terezina M.; Rajfur, Zenon; Jacobson, Ken

    2007-01-01

    The substrate-dependent movement of apicomplexan parasites such as Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain, and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. For the interaction of this complex with F-actin to result in net parasite movement, it is necessary that the myosin be immobilized with respect to the parasite and the actin with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not seem to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex, however, does seem to require cholesterol. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens such as Plasmodium and possibly to other eukaryotes. PMID:17538016

  9. Disentangling perceptual and motor components in inhibition of return.

    PubMed

    Zhou, Bin

    2008-08-01

    Following an abrupt onset of a peripheral stimulus (a cue), the response to a visual target is faster when the target appears at the cued position than when it appears at other positions. However, if the stimulus onset asynchrony (SOA) is longer than approximately 300 ms, the response to the target is slower at the cued position than that at other positions. This phenomenon of a longer response time to cued targets is called "inhibition of return" (IOR). Previous hypotheses propose contributions of both response inhibition and attentional inhibition at cued position to IOR, and suggest that responding to the cue can eliminate the component of response inhibition. The current study uses tasks either executing or withholding response to the cue to investigate the relative contributions of response and attention components to IOR. A condition with bilateral display of the cue is also chosen as a control condition, and eight different SOAs between 1,000 and 2,750 ms are tested. Compared to the control condition, response delay to the target at a cued position is eliminated by responding to the cue, and a response advantage to the target at an uncued position is not affected by responding to the cue. Furthermore, both response delay at a cued position and response advantage at an uncued position decrease with SOA in the time window tested in these experiments. The results reported here indicate a dominant response inhibition at a cued position and a primary attentional allocation at an uncued position for IOR. Nonsignificant perceptual/attentional suppression at a cued position is argued to be a benefit for visual detection in a changing world.

  10. Event-related desynchronization reflects downregulation of intracortical inhibition in human primary motor cortex.

    PubMed

    Takemi, Mitsuaki; Masakado, Yoshihisa; Liu, Meigen; Ushiba, Junichi

    2013-09-01

    There is increasing interest in electroencephalogram (EEG)-based brain-computer interface (BCI) as a tool for rehabilitation of upper limb motor functions in hemiplegic stroke patients. This type of BCI often exploits mu and beta oscillations in EEG recorded over the sensorimotor areas, and their event-related desynchronization (ERD) following motor imagery is believed to represent increased sensorimotor cortex excitability. However, it remains unclear whether the sensorimotor cortex excitability is actually correlated with ERD. Thus we assessed the association of ERD with primary motor cortex (M1) excitability during motor imagery of right wrist movement. M1 excitability was tested by motor evoked potentials (MEPs), short-interval intracortical inhibition (SICI), and intracortical facilitation (ICF) with transcranial magnetic stimulation (TMS). Twenty healthy participants were recruited. The participants performed 7 s of rest followed by 5 s of motor imagery and received online visual feedback of the ERD magnitude of the contralateral hand M1 while performing the motor imagery task. TMS was applied to the right hand M1 when ERD exceeded predetermined thresholds during motor imagery. MEP amplitudes, SICI, and ICF were recorded from the agonist muscle of the imagined hand movement. Results showed that the large ERD during wrist motor imagery was associated with significantly increased MEP amplitudes and reduced SICI but no significant changes in ICF. Thus ERD magnitude during wrist motor imagery represents M1 excitability. This study provides electrophysiological evidence that a motor imagery task involving ERD may induce changes in corticospinal excitability similar to changes accompanying actual movements.

  11. On the kinetics that moves Myosin V

    NASA Astrophysics Data System (ADS)

    Maes, Christian; O'Kelly de Galway, Winny

    2015-10-01

    Molecular motor proteins such as Myosin V, Dynein or Kinesin are no ratchets, at least not with a flashing asymmetric potential; the crucial asymmetry is in the dynamical activity. We make that explicit in terms of a simple Markov model, emphasizing the kinetic (and non-thermodynamic) aspects of stochastic transport. The analysis shows the presence of a fluctuation symmetry in that part of the dynamical activity which is antisymmetric under reversal of trailing and leading head of the motor. The direction of the motor motion is determined by it.

  12. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats.

    PubMed

    Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session. PMID:27314672

  13. Protein Synthesis Inhibition in the Peri-Infarct Cortex Slows Motor Recovery in Rats

    PubMed Central

    Schubring-Giese, Maximilian; Leemburg, Susan; Luft, Andreas Rüdiger; Hosp, Jonas Aurel

    2016-01-01

    Neuroplasticity and reorganization of brain motor networks are thought to enable recovery of motor function after ischemic stroke. Especially in the cortex surrounding the ischemic scar (i.e., peri-infarct cortex), evidence for lasting reorganization has been found at the level of neurons and networks. This reorganization depends on expression of specific genes and subsequent protein synthesis. To test the functional relevance of the peri-infarct cortex for recovery we assessed the effect of protein synthesis inhibition within this region after experimental stroke. Long-Evans rats were trained to perform a skilled-reaching task (SRT) until they reached plateau performance. A photothrombotic stroke was induced in the forelimb representation of the primary motor cortex (M1) contralateral to the trained paw. The SRT was re-trained after stroke while the protein synthesis inhibitor anisomycin (ANI) or saline were injected into the peri-infarct cortex through implanted cannulas. ANI injections reduced protein synthesis within the peri-infarct cortex by 69% and significantly impaired recovery of reaching performance through re-training. Improvement of motor performance within a single training session remained intact, while improvement between training sessions was impaired. ANI injections did not affect infarct size. Thus, protein synthesis inhibition within the peri-infarct cortex impairs recovery of motor deficits after ischemic stroke by interfering with consolidation of motor memory between training sessions but not short-term improvements within one session. PMID:27314672

  14. Model of Rho-Mediated Myosin Recruitment to the Cleavage Furrow during Cytokinesis

    NASA Astrophysics Data System (ADS)

    Veksler, Alexander; Vavylonis, Dimitrios

    2010-03-01

    The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During cytokinesis, the myosin attached to the cell's cortex progressively disassembles at the flanking regions and concentrates in the equator [1]. This recruitment depends on myosin motor activity and activation by Rho proteins. Central spindle and astral microtubules establish a spatial pattern of differential Rho activity [2]. We propose a reaction-diffusion model for the dynamics of myosin and Rho proteins during cytokinesis. In the model, the mitotic spindle activates Rho at the equator. Active Rho promotes, in a switch-like manner, myosin assembly into cortical minifilaments. Mechanical stress by cortical myosin causes disassembly of myosin minifilaments and deactivates Rho. Our results explain both the recruitment of myosin to the cleavage furrow and the observed damped myosin oscillations in the cell's flanking regions [1]. Spatial extent, period and decay rate of myosin oscillations are calculated. Various regimes of myosin recruitment are predicted. [1] Zhou & Wang, Mol. Biol. Cell 19:318 (2008) [2] Murthy & Wadsworth, J. Cell Sci. 121:2350 (2008)

  15. Nucleo-olivary inhibition balances the interaction between the reactive and adaptive layers in motor control.

    PubMed

    Herreros, Ivan; Verschure, Paul F M J

    2013-11-01

    In the acquisition of adaptive motor reflexes to aversive stimuli, the cerebellar output fulfills a double purpose: it controls a motor response and it relays a sensory prediction. However, the question of how these two apparently incompatible goals might be achieved by the same cerebellar area remains open. Here we propose a solution where the inhibition of the Inferior Olive (IO) by the cerebellar Deep Nuclei (DN) translates the motor command signal into a sensory prediction allowing a single cerebellar area to simultaneously tackle both aspects of the problem: execution and prediction. We demonstrate that having a graded error signal, the gain of the Nucleo-Olivary Inhibition (NOI) balances the generation of the response between the cerebellar and the reflexive controllers or, in other words, between the adaptive and the reactive layers of behavior. Moreover, we show that the resulting system is fully autonomous and can either acquire or erase adaptive responses according to their utility.

  16. 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

  17. Force-producing ADP state of myosin bound to actin

    PubMed Central

    Wulf, Sarah F.; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G.; Pylypenko, Olena; Sweeney, H. Lee; Houdusse, Anne M.; Schröder, Rasmus R.

    2016-01-01

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V. PMID:26976594

  18. Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster.

    PubMed

    Heissler, Sarah M; Chinthalapudi, Krishna; Sellers, James R

    2015-04-01

    Nonmuscle myosin-2 is the primary enzyme complex powering contractility of the F-actin cytoskeleton in the model organism Drosophila. Despite myosin's essential function in fly development and homeostasis, its kinetic features remain elusive. The purpose of this in vitro study is a detailed steady-state and presteady-state kinetic characterization of the Drosophila nonmuscle myosin-2 motor domain. Kinetic features are a slow steady-state ATPase activity, high affinities for F-actin and ADP, and a low duty ratio. Comparative analysis of the overall enzymatic signatures across the nonmuscle myosin-2 complement from model organisms indicates that the Drosophila protein resembles nonmuscle myosin-2s from metazoa rather than protozoa, though modulatory aspects of myosin motor function are distinct. Drosophila nonmuscle myosin-2 is uniquely insensitive toward blebbistatin, a commonly used myosin-2 inhibitor. An in silico modeling approach together with kinetic studies indicate that the nonconsensus amino acid Met466 in the Drosophila nonmuscle myosin-2 active-site loop switch-2 acts as blebbistatin desensitizer. Introduction of the M466I mutation sensitized the protein for blebbistatin, resulting in a half-maximal inhibitory concentration of 36.3 ± 4.1 µM. Together, these data show that Drosophila nonmuscle myosin-2 is a bona fide molecular motor and establish an important link between switch-2 and blebbistatin sensitivity.

  19. Tuning myosin-driven sorting on cellular actin networks

    PubMed Central

    Hariadi, Rizal F; Sommese, Ruth F; Sivaramakrishnan, Sivaraj

    2015-01-01

    Myosin V and VI are antagonistic motors that cohabit membrane vesicles in cells. A systematic study of their collective function, however, is lacking and forms the focus of this study. We functionally reconstitute a two-dimensional actin-myosin interface using myosin V and VI precisely patterned on DNA nanostructures, in combination with a model keratocyte actin meshwork. While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm. This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold. Pairing computation with experimental observations suggests that the ratio of motor stall forces is a key determinant of the observed competitive outcomes. Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type. DOI: http://dx.doi.org/10.7554/eLife.05472.001 PMID:25738229

  20. An identified glutamatergic interneuron patterns feeding motor activity via both excitation and inhibition.

    PubMed

    Quinlan, E M; Gregory, K; Murphy, A D

    1995-03-01

    1. Previously we demonstrated that glutamate is an important neurotransmitter in the CNS of Helisoma. Exogenous glutamate applied to the buccal ganglia mimicked both the excitatory and inhibitory effects of subunit 2 (S2) of the tripartite central pattern generator (CPG) on S2 postsynaptic motor neurons. Here we identify buccal interneuron B2 as an S2 interneuron by utilizing a combination of electrophysiology, pharmacology, and intracellular staining. In addition, neurons that were electrophysiologically and morphologically characterized as neuron B2 demonstrated antiglutamate immunoreactivity, suggesting that neuron B2 is a source of endogenous glutamate in the buccal ganglia. 2. Depolarization of neuron B2 evoked excitatory postsynaptic potentials in motor neurons excited by S2. The excitatory effects of B2 depolarization and S2 activation were reversibly antagonized by the ionotropic glutamate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione, similar to the antagonism shown previously for application of exogenous glutamate. Depolarization of neuron B2 also evoked inhibitory postsynaptic potentials in motor neurons inhibited by S2. When such motor neurons were maintained in isolated cell culture, application of exogenous glutamate produced a direct hyperpolarization of the membrane potential. 3. The activity of neuron B2 is necessary for the production of the standard pattern of buccal motor neuron activity, which underlies functional feeding movements. The subunits of the tripartite buccal CPG must be active in the temporal sequence S1-S2-S3 to produce the standard feeding pattern. Rhythmic inhibition from neuron B2 terminated activity in S1 postsynaptic motor neurons and entrained the frequency of activity in S3 postsynaptic motor neurons. Hyperpolarization of neuron B2 disrupted the production of the standard motor pattern by eliminating S2 postsynaptic potentials in identified buccal motor neurons, thereby prolonging S1 activity and disrupting S3

  1. Globular tail of myosin-V is bound to vamp/synaptobrevin.

    PubMed

    Ohyama, A; Komiya, Y; Igarashi, M

    2001-02-01

    VAMP/synaptobrevin is one of a number of v-SNAREs involved in vesicular fusion events in neurons. In a previous report, VAMP was shown to form a complex with synaptophysin and myosin V, a motor protein based on the F-actin, and that myosin V was then released from the complex in a Ca(2+)-dependent manner. Here, we found that VAMP alone is bound to myosin V in a Ca(2+)-independent manner, and determined that the globular tail domain of myosin V is its binding site. The syntaxin-VAMP-myosin V formed in the presence of Ca(2+)/calmodulin (CaM). In the absence of CaM, only syntaxin-VAMP, or VAMP-myosin V complex was formed. Our results suggest that VAMP acts as a myosin V receptor on the vesicles and regulates formation of the complex.

  2. Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement

    PubMed Central

    Fabian, Lacramioara; Troscianczuk, Joanna; Forer, Arthur

    2007-01-01

    Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise. PMID:17381845

  3. Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement.

    PubMed

    Fabian, Lacramioara; Troscianczuk, Joanna; Forer, Arthur

    2007-01-01

    Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise. PMID:17381845

  4. Motor Inhibition Affects the Speed But Not Accuracy of Aimed Limb Movements in an Insect

    PubMed Central

    Calas-List, Delphine; Clare, Anthony J.; Komissarova, Alexandra; Nielsen, Thomas A.

    2014-01-01

    When reaching toward a target, human subjects use slower movements to achieve higher accuracy, and this can be accompanied by increased limb impedance (stiffness, viscosity) that stabilizes movements against motor noise and external perturbation. In arthropods, the activity of common inhibitory motor neurons influences limb impedance, so we hypothesized that this might provide a mechanism for speed and accuracy control of aimed movements in insects. We recorded simultaneously from excitatory leg motor neurons and from an identified common inhibitory motor neuron (CI1) in locusts that performed natural aimed scratching movements. We related limb movement kinematics to recorded motor activity and demonstrate that imposed alterations in the activity of CI1 influenced these kinematics. We manipulated the activity of CI1 by injecting depolarizing or hyperpolarizing current or killing the cell using laser photoablation. Naturally higher levels of inhibitory activity accompanied faster movements. Experimentally biasing the firing rate downward, or stopping firing completely, led to slower movements mediated by changes at several joints of the limb. Despite this, we found no effect on overall movement accuracy. We conclude that inhibitory modulation of joint stiffness has effects across most of the working range of the insect limb, with a pronounced effect on the overall velocity of natural movements independent of their accuracy. Passive joint forces that are greatest at extreme joint angles may enhance accuracy and are not affected by motor inhibition. PMID:24872556

  5. 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.

  6. Review: The ATPase mechanism of myosin and actomyosin.

    PubMed

    Geeves, Michael A

    2016-08-01

    Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 483-491, 2016. PMID:27061920

  7. Modulation of interhemispheric inhibition by volitional motor activity: an ipsilateral silent period study.

    PubMed

    Giovannelli, Fabio; Borgheresi, Alessandra; Balestrieri, Fabrizio; Zaccara, Gaetano; Viggiano, Maria Pia; Cincotta, Massimo; Ziemann, Ulf

    2009-11-15

    Brief interruption of voluntary EMG in a hand muscle by focal transcranial magnetic stimulation (TMS) of the ipsilateral primary motor cortex (M1), the so-called ipsilateral silent period (ISP), is a measure of interhemispheric motor inhibition. However, little is known about how volitional motor activity would modulate the ISP. Here we tested in 30 healthy adults to what extent and under what conditions voluntary activation of the stimulated right M1 by moving the left hand strengthens interhemispheric inhibition as indexed by an enhancement of the ISP area in the maximally contracting right first dorsal interosseous (FDI). Left index finger abduction, already at low levels of contraction, significantly enhanced the ISP compared to left hand at rest. Even imagination of left index finger movement enhanced the ISP compared to rest or mental calculation. This enhancement occurred in the absence of motor-evoked potential amplitude modulation in the left FDI, thus excluding a non-specific contribution from an increase in right M1 corticospinal excitability. Contraction of the left extensor indicis, but not contraction of more proximal left upper limb or left or right lower limb muscles also enhanced the ISP. A reaction time experiment showed that the ISP enhancement developed at a late stage of movement preparation just before or at movement onset. Interhemispheric inhibition of the motor-evoked potential as tested by a bifocal paired-pulse TMS protocol and thought to be mediated via a neuronal circuit different to the ISP was not enhanced when tested under identical motor task conditions. Finally, ISP enhancement by contraction of the left FDI correlated inversely with EMG mirror activity in the right FDI during phasic abductions of the left index finger. Our findings strongly suggest that voluntary M1 activation by real or imagined movement of the contralateral hand increases interhemispheric motor inhibition of the opposite M1. This phenomenon shows substantial

  8. Long-latency TMS-evoked potentials during motor execution and inhibition.

    PubMed

    Yamanaka, Kentaro; Kadota, Hiroshi; Nozaki, Daichi

    2013-01-01

    Transcranial magnetic stimulation (TMS) has often been used in conjunction with electroencephalography (EEG), which is effective for the direct demonstration of cortical reactivity and corticocortical connectivity during cognitive tasks through the spatio-temporal pattern of long-latency TMS-evoked potentials (TEPs). However, it remains unclear what pattern is associated with the inhibition of a planned motor response. Therefore, we performed TMS-EEG recording during a go/stop task, in which participants were instructed to click a computer mouse with a right index finger when an indicator that was moving with a constant velocity reached a target (go trial) or to avoid the click when the indicator randomly stopped just before it reached the target (stop trial). Single-pulse TMS to the left (contralateral) or right (ipsilateral) motor cortex was applied 500 ms before or just at the target time. TEPs related to motor execution and inhibition were obtained by subtractions between averaged EEG waveforms with and without TMS. As a result, in TEPs induced by both contralateral and ipsilateral TMS, small oscillations were followed by a prominent negative deflection around the TMS site peaking at approximately 100 ms post-TMS (N100), and a less pronounced later positive component (LPC) over the broad areas that was centered at the midline-central site in both go and stop trials. However, compared to the pattern in go and stop trials with TMS at 500 ms before the target time, N100 and LPC were differently modulated in the go and stop trials with TMS just at the target time. The amplitudes of both N100 and LPC decreased in go trials, while the amplitude of LPC decreased and the latency of LPC was delayed in both go and stop trials. These results suggested that TMS-induced neuronal reactions in the motor cortex and subsequent their propagation to surrounding cortical areas might change functionally according to task demand when executing and inhibiting a motor response.

  9. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    PubMed

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  10. 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. PMID:26707799

  11. Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures.

    PubMed

    Jaenisch, Nadine; Liebmann, Lutz; Guenther, Madlen; Hübner, Christian A; Frahm, Christiane; Witte, Otto W

    2016-01-01

    Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4β3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors. PMID:27188341

  12. Reduced tonic inhibition after stroke promotes motor performance and epileptic seizures

    PubMed Central

    Jaenisch, Nadine; Liebmann, Lutz; Guenther, Madlen; Hübner, Christian A.; Frahm, Christiane; Witte, Otto W.

    2016-01-01

    Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4β3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors. PMID:27188341

  13. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va.

    PubMed

    Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P

    2012-01-01

    Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

  14. Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va

    PubMed Central

    Azevedo, Elisama; Oliveira, Leandro Teixeira; Castro Lima, Ana Karina; Terra, Rodrigo; Dutra, Patrícia Maria Lourenço; Salerno, Verônica P.

    2012-01-01

    Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections. PMID:22792440

  15. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  16. 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

    2010-08-01

    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.

  17. My oh my(osin): Insights into how auditory hair cells count, measure, and shape.

    PubMed

    Pollock, Lana M; Chou, Shih-Wei; McDermott, Brian M

    2016-01-18

    The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle's morphology and hearing.

  18. My oh my(osin): Insights into how auditory hair cells count, measure, and shape

    PubMed Central

    Pollock, Lana M.; Chou, Shih-Wei

    2016-01-01

    The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle’s morphology and hearing. PMID:26754648

  19. Still and rotating myosin clusters determine cytokinetic ring constriction.

    PubMed

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel

    2016-07-01

    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.

  20. Still and rotating myosin clusters determine cytokinetic ring constriction.

    PubMed

    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

  1. Functional roles for myosin 1c in cellular signaling pathways

    PubMed Central

    Bond, Lisa M.; Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Cellular signaling pathways underlie the transfer of information throughout the cell and to adjoining cells and so govern most critical cellular functions. Increasing evidence points to the molecular motor myosin 1c as a prominent player in many signaling cascades, from the integrin-dependent signaling involved in cell migration to the signaling events underlying insulin resistance. Myosin 1c functions on these pathways both via an important role in regulating lipid raft recycling and also via direct involvement in signaling cascades. This review provides an overview of the functional involvement of myosin 1c in cellular signaling and discusses the possible potential for myosin 1c as a target for drug-based treatments for human diseases. PMID:23022959

  2. 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

  3. Myosin light chain kinase accelerates vesicle endocytosis at the calyx of Held synapse.

    PubMed

    Yue, Hai-Yuan; Xu, Jianhua

    2014-01-01

    Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca(2+) and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca(2+)/calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca(2+) channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca(2+)/calmodulin.

  4. 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.

  5. Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic β-cells

    PubMed Central

    Han, Young-Eun; Lim, Ajin; Park, Sun-Hyun; Chang, Sunghoe; Lee, Suk-Ho; Ho, Won-Kyung

    2015-01-01

    AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic β-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic β-cells. PMID:26471000

  6. Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

    PubMed Central

    Anderson, Tom W.; Vaughan, Andrew N.

    2008-01-01

    In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles. PMID:18799629

  7. Comparison of Motor Inhibition in Variants of the Instructed-Delay Choice Reaction Time Task.

    PubMed

    Quoilin, Caroline; Lambert, Julien; Jacob, Benvenuto; Klein, Pierre-Alexandre; Duque, Julie

    2016-01-01

    Using instructed-delay choice reaction time (RT) paradigms, many previous studies have shown that the motor system is transiently inhibited during response preparation: motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) over the primary motor cortex are typically suppressed during the delay period. This effect has been observed in both selected and non-selected effectors, although MEP changes in selected effectors have been more inconsistent across task versions. Here, we compared changes in MEP amplitudes in three different variants of an instructed-delay choice RT task. All variants required participants to choose between left and right index finger movements but the responses were either provided "in the air" (Variant 1), on a regular keyboard (Variant 2), or on a response device designed to control from premature responses (Variant 3). The task variants also differed according to the visual layout (more concrete in Variant 3) and depending on whether participants received a feedback of their performance (absent in Variant 1). Behavior was globally comparable between the three variants of the task although the propensity to respond prematurely was highest in Variant 2 and lowest in Variant 3. MEPs elicited in a non-selected hand were similarly suppressed in the three variants of the task. However, significant differences emerged when considering MEPs elicited in the selected hand: these MEPs were suppressed in Variants 1 and 3 whereas they were often facilitated in Variant 2, especially in the right dominant hand. In conclusion, MEPs elicited in selected muscles seem to be more sensitive to small variations to the task design than those recorded in non-selected effectors, probably because they reflect a complex combination of inhibitory and facilitatory influences on the motor output system. Finally, the use of a standard keyboard seems to be particularly inappropriate because it encourages participants to respond promptly with no

  8. Associations between regional brain physiology and trait impulsivity, motor inhibition, and impaired control over drinking

    PubMed Central

    Weafer, Jessica; Dzemidzic, Mario; Eiler, William; Oberlin, Brandon G.; Wang, Yang; Kareken, David A.

    2015-01-01

    Trait impulsivity and poor inhibitory control are well-established risk factors for alcohol misuse, yet little is known about the associated neurobiological endophenotypes. Here we examined correlations among brain physiology and self-reported trait impulsive behavior, impaired control over drinking, and a behavioral measure of response inhibition. A sample of healthy drinkers (n=117) completed a pulsed arterial spin labeling (PASL) scan to quantify resting regional cerebral blood flow (rCBF), and measures of self-reported impulsivity (Eysenck I7 Impulsivity scale) and impaired control over drinking. A subset of subjects (n=40) performed a stop signal task during blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging to assess brain regions involved in response inhibition. Eysenck I7 scores were inversely related to blood flow in the right precentral gyrus. Significant BOLD activation during response inhibition occurred in an overlapping right frontal motor/premotor region. Moreover, impaired control over drinking was associated with reduced BOLD response in the same region. These findings suggest that impulsive personality and impaired control over drinking are associated with brain physiology in areas implicated in response inhibition. This is consistent with the idea that difficulty controlling behavior is due in part to impairment in motor restraint systems. PMID:26065376

  9. Plasmodium falciparum myosins: transcription and translation during asexual parasite development.

    PubMed

    Chaparro-Olaya, Jacqueline; Margos, Gabriele; Coles, Deborah J; Dluzewski, Anton R; Mitchell, Graham H; Wasserman, Moisés M; Pinder, Jennifer C

    2005-04-01

    Six myosins genes are now annotated in the Plasmodium falciparum Genome Project. Malaria myosins have been named alphabetically; accordingly, we refer to the two latest additions as Pfmyo-E and Pfmyo-F. Both new myosins contain regions characteristic of the functional motor domain of "true" myosins and, unusually for P. falciparum myosins, Pfmyo-F encodes two consensus IQ light chain-binding motifs. Phylogenetic analysis of the 17 currently known apicomplexan myosins together with one representative of each myosin class clusters all but one of the apicomplexan sequences together in Class XIV. This refines the earlier definition of the Class XIV Subclasses XIVa and XIVb. RT-PCR on blood stage parasite mRNA amplifies a specific product for all six myosins and each shows developmentally regulated transcription. Thus: Pfmyo-A and Pfmyo-B genes are transcribed throughout development; Pfmyo-C is predominant in trophozoites; Pfmyo-D occurs in trophozoites and schizonts; Pfmyo-E though barely present in earlier stages is abundant in schizonts; Pfmyo-F increases steadily throughout development and maturation. It is known that Pfmyo-A and Pfmyo-B are synthesised during late schizogony and we now show that Pfmyo-D expression is also temporally regulated to late trophozoites and schizonts where it distributes close to segregating nuclei. Thus, in asexual stages myosin synthesis does not always parallel transcript accumulation, showing that translation is also regulated. The implication is that the mRNAs are either subjected to turnover, synthesised and degraded, or that they are sequestered in an inactivate form until required for protein synthesis.

  10. Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth.

    PubMed

    Takeshita, Norio; Yamashita, Shuichi; Ohta, Akinori; Horiuchi, Hiroyuki

    2006-03-01

    The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.

  11. Aging and motor inhibition: a converging perspective provided by brain stimulation and imaging approaches.

    PubMed

    Levin, Oron; Fujiyama, Hakuei; Boisgontier, Matthieu P; Swinnen, Stephan P; Summers, Jeffery J

    2014-06-01

    The ability to inhibit actions, one of the hallmarks of human motor control, appears to decline with advancing age. Evidence for a link between changes in inhibitory functions and poor motor performance in healthy older adults has recently become available with transcranial magnetic stimulation (TMS). Overall, these studies indicate that the capacity to modulate intracortical (ICI) and interhemispheric (IHI) inhibition is preserved in high-performing older individuals. In contrast, older individuals exhibiting motor slowing and a declined ability to coordinate movement appear to show a reduced capability to modulate GABA-mediated inhibitory processes. As a decline in the integrity of the GABA-ergic inhibitory processes may emerge due to age-related loss of white and gray matter, a promising direction for future research would be to correlate individual differences in structural and/or functional integrity of principal brain networks with observed changes in inhibitory processes within cortico-cortical, interhemispheric, and/or corticospinal pathways. Finally, we underscore the possible links between reduced inhibitory functions and age-related changes in brain activation patterns.

  12. Cutaneous afferent input does not modulate motor intracortical inhibition in ageing men.

    PubMed

    Smith, Ashleigh E; Ridding, Michael C; Higgins, Ryan D; Wittert, Gary A; Pitcher, Julia B

    2011-11-01

    Afferent input has been shown to be a powerful modulator of cortical inhibition. Such modulation is likely to be important for the control of ongoing movement, but may also play a role in facilitating neuroplastic reorganisation. Human motor control and neuroplasticity both decline with ageing, whereas the efficacy of short-interval intracortical inhibition (SICI) appears not to. We examined if ageing alters the efficacy of afferent modulation of SICI. Previously, electrical cutaneous stimulation of a finger has been shown to reduce SICI in the motor cortices of young adults. Paired-pulse transcranial magnetic stimulation was used to assess SICI in the cortical representation of the first dorsal interosseous muscle. SICI was assessed separately under two conditions: with and without prior afferent input from electrical cutaneous stimulation of the index finger. Fifteen 'young' (20.1 ± 2.1 years) and 15 'old' male humans (65.5 ± 3.9 years) were studied. SICI did not differ when young and old males were compared. However, when preceded by electrical cutaneous finger stimulation, SICI was reduced in young men but not old men. Reflex testing indicated preservation of the afferent volley to the cortex. These findings suggest that a contributing factor in the decline of motor function, and possibly neuroplasticity, with ageing is loss of SICI modulation, probably due to altered cortical sensorimotor integration of afferent input.

  13. The neurophysiology of response competition: motor cortex activation and inhibition following subliminal response priming.

    PubMed

    Praamstra, Peter; Seiss, Ellen

    2005-03-01

    Some widely used tasks in cognitive neuroscience depend on the induction of a response conflict between choice alternatives, involving partial activation of the incorrect response before the correct response is emitted. Although such ''conflict tasks'' are often used to investigate frontal-lobe-based conflict-monitoring processes, it is not known how response competition evolves in the motor cortex. To investigate the dynamics of motor cortex activation during response competition, we used a subliminal priming task that induced response competition while bypassing pre-response stage processing conflict. Analyses of movement-related EEG potentials supported an interaction between competing responses characterized by reciprocal inhibition. Inhibitory interactions between response channels contribute to the resolution of response conflict. However, the reciprocal inhibition at motor cortex level seemed to operate independent of higher level conflict-monitoring processes, which were relatively insensitive to response conflict induced by subliminal priming. These results elucidate how response conflict causes interference as well as the conditions under which frontal-lobe-based interference control processes are engaged.

  14. Automatic ultrarapid activation and inhibition of cortical motor systems in spoken word comprehension

    PubMed Central

    Shtyrov, Yury; Butorina, Anna; Nikolaeva, Anastasia; Stroganova, Tatiana

    2014-01-01

    To address the hotly debated question of motor system involvement in language comprehension, we recorded neuromagnetic responses elicited in the human brain by unattended action-related spoken verbs and nouns and scrutinized their timecourse and neuroanatomical substrates. We found that already very early on, from ∼80 ms after disambiguation point when the words could be identified from the available acoustic information, both verbs and nouns produced characteristic somatotopic activations in the motor strip, with words related to different body parts activating the corresponding body representations. Strikingly, along with this category-specific activation, we observed suppression of motor-cortex activation by competitor words with incompatible semantics, documenting operation of the neurophysiological principles of lateral/surround inhibition in neural word processing. The extremely early onset of these activations and deactivations, their emergence in the absence of attention, and their similar presence for words of different lexical classes strongly suggest automatic involvement of motor-specific circuits in the perception of action-related language. PMID:24753617

  15. Automatic ultrarapid activation and inhibition of cortical motor systems in spoken word comprehension.

    PubMed

    Shtyrov, Yury; Butorina, Anna; Nikolaeva, Anastasia; Stroganova, Tatiana

    2014-05-01

    To address the hotly debated question of motor system involvement in language comprehension, we recorded neuromagnetic responses elicited in the human brain by unattended action-related spoken verbs and nouns and scrutinized their timecourse and neuroanatomical substrates. We found that already very early on, from ∼80 ms after disambiguation point when the words could be identified from the available acoustic information, both verbs and nouns produced characteristic somatotopic activations in the motor strip, with words related to different body parts activating the corresponding body representations. Strikingly, along with this category-specific activation, we observed suppression of motor-cortex activation by competitor words with incompatible semantics, documenting operation of the neurophysiological principles of lateral/surround inhibition in neural word processing. The extremely early onset of these activations and deactivations, their emergence in the absence of attention, and their similar presence for words of different lexical classes strongly suggest automatic involvement of motor-specific circuits in the perception of action-related language.

  16. Mirror illusion reduces motor cortical inhibition in the ipsilateral primary motor cortex during forceful unilateral muscle contractions.

    PubMed

    Zult, Tjerk; Goodall, Stuart; Thomas, Kevin; Hortobágyi, Tibor; Howatson, Glyn

    2015-04-01

    Forceful, unilateral contractions modulate corticomotor paths targeting the resting, contralateral hand. However, it is unknown whether mirror-viewing of a slowly moving but forcefully contracting hand would additionally affect these paths. Here we examined corticospinal excitability and short-interval intracortical inhibition (SICI) of the right-ipsilateral primary motor cortex (M1) in healthy young adults under no-mirror and mirror conditions at rest and during right wrist flexion at 60% maximal voluntary contraction (MVC). During the no-mirror conditions neither hand was visible, whereas in the mirror conditions participants looked at the right hand's reflection in the mirror. Corticospinal excitability increased during contractions in the left flexor carpi radialis (FCR) (contraction 0.41 mV vs. rest 0.21 mV) and extensor carpi radialis (ECR) (contraction 0.56 mV vs. rest 0.39 mV), but there was no mirror effect (FCR: P = 0.743, ηp (2) = 0.005; ECR: P = 0.712, ηp (2) = 0.005). However, mirror-viewing of the contracting and moving wrist attenuated SICI relative to test pulse in the left FCR by ∼9% compared with the other conditions (P < 0.05, d ≥ 0.62). Electromyographic activity in the resting left hand prior to stimulation was not affected by the mirror (FCR: P = 0.255, ηp (2) = 0.049; ECR: P = 0.343, ηp (2) = 0.035) but increased twofold during contractions. Thus viewing the moving hand in the mirror and not just the mirror image of the nonmoving hand seems to affect motor cortical inhibitory networks in the M1 associated with the mirror image. Future studies should determine whether the use of a mirror could increase interlimb transfer produced by cross-education, especially in patient groups with unilateral orthopedic and neurological conditions.

  17. Mirror illusion reduces motor cortical inhibition in the ipsilateral primary motor cortex during forceful unilateral muscle contractions.

    PubMed

    Zult, Tjerk; Goodall, Stuart; Thomas, Kevin; Hortobágyi, Tibor; Howatson, Glyn

    2015-04-01

    Forceful, unilateral contractions modulate corticomotor paths targeting the resting, contralateral hand. However, it is unknown whether mirror-viewing of a slowly moving but forcefully contracting hand would additionally affect these paths. Here we examined corticospinal excitability and short-interval intracortical inhibition (SICI) of the right-ipsilateral primary motor cortex (M1) in healthy young adults under no-mirror and mirror conditions at rest and during right wrist flexion at 60% maximal voluntary contraction (MVC). During the no-mirror conditions neither hand was visible, whereas in the mirror conditions participants looked at the right hand's reflection in the mirror. Corticospinal excitability increased during contractions in the left flexor carpi radialis (FCR) (contraction 0.41 mV vs. rest 0.21 mV) and extensor carpi radialis (ECR) (contraction 0.56 mV vs. rest 0.39 mV), but there was no mirror effect (FCR: P = 0.743, ηp (2) = 0.005; ECR: P = 0.712, ηp (2) = 0.005). However, mirror-viewing of the contracting and moving wrist attenuated SICI relative to test pulse in the left FCR by ∼9% compared with the other conditions (P < 0.05, d ≥ 0.62). Electromyographic activity in the resting left hand prior to stimulation was not affected by the mirror (FCR: P = 0.255, ηp (2) = 0.049; ECR: P = 0.343, ηp (2) = 0.035) but increased twofold during contractions. Thus viewing the moving hand in the mirror and not just the mirror image of the nonmoving hand seems to affect motor cortical inhibitory networks in the M1 associated with the mirror image. Future studies should determine whether the use of a mirror could increase interlimb transfer produced by cross-education, especially in patient groups with unilateral orthopedic and neurological conditions. PMID:25632077

  18. 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

  19. Thirteen is enough: the myosins of Dictyostelium discoideum and their light chains

    PubMed Central

    Kollmar, Martin

    2006-01-01

    Background Dictyostelium discoideum is one of the most famous model organisms for studying motile processes like cell movement, organelle transport, cytokinesis, and endocytosis. Members of the myosin superfamily, that move on actin filaments and power many of these tasks, are tripartite proteins consisting of a conserved catalytic domain followed by the neck region consisting of a different number of so-called IQ motifs for binding of light chains. The tails contain functional motifs that are responsible for the accomplishment of the different tasks in the cell. Unicellular organisms like yeasts contain three to five myosins while vertebrates express over 40 different myosin genes. Recently, the question has been raised how many myosins a simple multicellular organism like Dictyostelium would need to accomplish all the different motility-related tasks. Results The analysis of the Dictyostelium genome revealed thirteen myosins of which three have not been described before. The phylogenetic analysis of the motor domains of the new myosins placed Myo1F to the class-I myosins and Myo5A to the class-V myosins. The third new myosin, an orphan myosin, has been named MyoG. It contains an N-terminal extension of over 400 residues, and a tail consisting of four IQ motifs and two MyTH4/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) tandem domains that are separated by a long region containing an SH3 (src homology 3) domain. In contrast to previous analyses, an extensive comparison with 126 class-VII, class-X, class-XV, and class-XXII myosins now showed that MyoI does not group into any of these classes and should not be used as a model for class-VII myosins. The search for calmodulin related proteins revealed two further potential myosin light chains. One is a close homolog of the two EF-hand motifs containing MlcB, and the other, CBP14, phylogenetically groups to the ELC/RLC/calmodulin (essential light chain/regulatory light chain) branch of the tree

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. Functional networks of motor inhibition in conversion disorder patients and feigning subjects.

    PubMed

    Hassa, Thomas; de Jel, Esther; Tuescher, Oliver; Schmidt, Roger; Schoenfeld, Mircea Ariel

    2016-01-01

    The neural correlates of motor inhibition leading to paresis in conversion disorder are not well known. The key question is whether they are different of those of normal subjects feigning the symptoms. Thirteen conversion disorder patients with hemiparesis and twelve healthy controls were investigated using functional magnetic resonance tomography under conditions of passive motor stimulation of the paretic/feigned paretic and the non-paretic hand. Healthy controls were also investigated in a non-feigning condition. During passive movement of the affected right hand conversion disorder patients exhibited activations in the bilateral triangular part of the inferior frontal gyri (IFG), with a left side dominance compared to controls in non-feigning condition. Feigning controls revealed for the same condition a weak unilateral activation in the right triangular part of IFG and an activity decrease in frontal midline areas, which couldn't be observed in patients. The results suggest that motor inhibition in conversion disorder patients is mediated by the IFG that was also involved in inhibition processes in normal subjects. The activity pattern in feigning controls resembled that of conversion disorder patients but with a clear difference in the medial prefrontal cortex. Healthy controls showed decreased activity in this region during feigning compared to non-feigning conditions suggesting a reduced sense of self-agency during feigning. Remarkably, no activity differences could be observed in medial prefrontal cortex for patients vs healthy controls in feigning or non-feigning conditions suggesting self-agency related activity in patients to be in between those of non-feigning and feigning healthy subjects. PMID:27330971

  5. Functional networks of motor inhibition in conversion disorder patients and feigning subjects.

    PubMed

    Hassa, Thomas; de Jel, Esther; Tuescher, Oliver; Schmidt, Roger; Schoenfeld, Mircea Ariel

    2016-01-01

    The neural correlates of motor inhibition leading to paresis in conversion disorder are not well known. The key question is whether they are different of those of normal subjects feigning the symptoms. Thirteen conversion disorder patients with hemiparesis and twelve healthy controls were investigated using functional magnetic resonance tomography under conditions of passive motor stimulation of the paretic/feigned paretic and the non-paretic hand. Healthy controls were also investigated in a non-feigning condition. During passive movement of the affected right hand conversion disorder patients exhibited activations in the bilateral triangular part of the inferior frontal gyri (IFG), with a left side dominance compared to controls in non-feigning condition. Feigning controls revealed for the same condition a weak unilateral activation in the right triangular part of IFG and an activity decrease in frontal midline areas, which couldn't be observed in patients. The results suggest that motor inhibition in conversion disorder patients is mediated by the IFG that was also involved in inhibition processes in normal subjects. The activity pattern in feigning controls resembled that of conversion disorder patients but with a clear difference in the medial prefrontal cortex. Healthy controls showed decreased activity in this region during feigning compared to non-feigning conditions suggesting a reduced sense of self-agency during feigning. Remarkably, no activity differences could be observed in medial prefrontal cortex for patients vs healthy controls in feigning or non-feigning conditions suggesting self-agency related activity in patients to be in between those of non-feigning and feigning healthy subjects.

  6. A Perspective on the Role of Myosins as Mechanosensors.

    PubMed

    Greenberg, Michael J; Arpağ, Göker; Tüzel, Erkan; Ostap, E Michael

    2016-06-21

    Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms. PMID:27332116

  7. Regulation of myosin II during cytokinesis in higher eukaryotes.

    PubMed

    Matsumura, Fumio

    2005-07-01

    Cellular myosin II is the principal motor responsible for cytokinesis. In higher eukaryotes, phosphorylation of the regulatory light chain (MLC) of myosin II is a primary means of activating myosin II and is known to be crucial for the execution of cell division. Because signals transmitted by the mitotic spindle coordinate key spatial and temporal aspects of cytokinesis, such signals should ultimately function to activate myosin II. Thus, it follows that identification of regulatory factors involved in MLC phosphorylation should elucidate the nature of spindle-derived regulatory signals and lead to a model for how they control cytokinesis. However, the identity of these upstream molecules remains elusive. This review (which is part of the Cytokinesis series) summarizes current views of the regulatory pathway controlling MLC phosphorylation and features four candidate molecules that are likely immediate upstream myosin regulators. I discuss proposed functions for MLCK, ROCK, citron kinase and myosin phosphatase during cytokinesis and consider the possibility of a link between these molecules and the signals transmitted by the mitotic spindle.

  8. Engineering myosins for long-range transport on actin filaments.

    PubMed

    Schindler, Tony D; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1).

  9. Engineering myosins for long-range transport on actin filaments

    NASA Astrophysics Data System (ADS)

    Schindler, Tony D.; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s-1.

  10. Engineering myosins for long-range transport on actin filaments.

    PubMed

    Schindler, Tony D; Chen, Lu; Lebel, Paul; Nakamura, Muneaki; Bryant, Zev

    2014-01-01

    Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1). PMID:24240432

  11. The mirror world of motor inhibition: the alien hand syndrome in chronic stroke.

    PubMed

    Brainin, M; Seiser, A; Matz, K

    2008-03-01

    of complex inhibitory motor programmes that have become isolated from normal motor planning, which usually suppresses them via the contralateral cortico-subcortical prefrontal circuits and the corpus callosum. Thus, the mirror world of complex motor inhibition becomes clinically visible in such patients.

  12. Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division.

    PubMed

    Silverman-Gavrila, Rosalind V; Forer, Arthur

    2003-06-01

    We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the

  13. Slow myosin in developing rat skeletal muscle

    PubMed Central

    1987-01-01

    Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC

  14. Comparison of Motor Inhibition in Variants of the Instructed-Delay Choice Reaction Time Task

    PubMed Central

    Quoilin, Caroline; Lambert, Julien; Jacob, Benvenuto; Klein, Pierre-Alexandre; Duque, Julie

    2016-01-01

    Using instructed-delay choice reaction time (RT) paradigms, many previous studies have shown that the motor system is transiently inhibited during response preparation: motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) over the primary motor cortex are typically suppressed during the delay period. This effect has been observed in both selected and non-selected effectors, although MEP changes in selected effectors have been more inconsistent across task versions. Here, we compared changes in MEP amplitudes in three different variants of an instructed-delay choice RT task. All variants required participants to choose between left and right index finger movements but the responses were either provided “in the air” (Variant 1), on a regular keyboard (Variant 2), or on a response device designed to control from premature responses (Variant 3). The task variants also differed according to the visual layout (more concrete in Variant 3) and depending on whether participants received a feedback of their performance (absent in Variant 1). Behavior was globally comparable between the three variants of the task although the propensity to respond prematurely was highest in Variant 2 and lowest in Variant 3. MEPs elicited in a non-selected hand were similarly suppressed in the three variants of the task. However, significant differences emerged when considering MEPs elicited in the selected hand: these MEPs were suppressed in Variants 1 and 3 whereas they were often facilitated in Variant 2, especially in the right dominant hand. In conclusion, MEPs elicited in selected muscles seem to be more sensitive to small variations to the task design than those recorded in non-selected effectors, probably because they reflect a complex combination of inhibitory and facilitatory influences on the motor output system. Finally, the use of a standard keyboard seems to be particularly inappropriate because it encourages participants to respond promptly

  15. Nigral proteasome inhibition in mice leads to motor and non-motor deficits and increased expression of Ser129 phosphorylated α-synuclein

    PubMed Central

    Bentea, Eduard; Van der Perren, Anke; Van Liefferinge, Joeri; El Arfani, Anissa; Albertini, Giulia; Demuyser, Thomas; Merckx, Ellen; Michotte, Yvette; Smolders, Ilse; Baekelandt, Veerle; Massie, Ann

    2015-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 μg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets. PMID:25873870

  16. Peripheral antagonistic action of trimebutine and kappa opioid substances on acoustic stress-induced gastric motor inhibition in dogs.

    PubMed

    Gué, M; Pascaud, X; Hondé, C; Junien, J L; Buéno, L

    1988-01-27

    The effects of intracerebroventricular (i.c.v.), intravenous (i.v.) and oral (p.o.) administration of trimebutine on the gastric motor inhibition induced by acoustic stress were investigated in fasted dogs fitted with strain-gauge transducers on the antrum and proximal jejunum. Started 40-50 min after the last migrating motor complex, a 1 h acoustic stress delayed by 111% the occurrence of the next gastric migrating motor complex without affecting the jejunal motor pattern. This inhibition of gastric migrating motor complex induced by acoustic stress was abolished by previous p.o. administration of trimebutine (1 mg/kg) but not by its i.v. (0.1 mg/kg) or i.c.v. (0.01 mg/kg) injection. The trimebutine blockade of gastric motor alterations induced by acoustic stress was suppressed after previous i.v. treatment with MR 2266 (0.3 mg/kg) but was unaffected by naloxone (0.3 mg/kg). Furthermore oral administration of U-50488H (10 micrograms/kg) and ethylketocyclazocine (10 micrograms/kg) respectively abolished and reduced the acoustic stress-induced delay of the occurrence of the gastric migrating motor complex. We concluded that trimebutine is able to antagonize the gastric motor disturbances induced in dogs by acoustic stress, probably by acting selectively on peripheral kappa receptors located in the wall of the proximal gut and directly stimulated from a mucosal site. PMID:2895010

  17. Crucial role for the LSP1-myosin1e bimolecular complex in the regulation of Fcγ receptor-driven phagocytosis.

    PubMed

    Maxeiner, Sebastian; Shi, Nian; Schalla, Carmen; Aydin, Guelcan; Hoss, Mareike; Vogel, Simon; Zenke, Martin; Sechi, Antonio S

    2015-05-01

    Actin cytoskeleton remodeling is fundamental for Fcγ receptor-driven phagocytosis. In this study, we find that the leukocyte-specific protein 1 (LSP1) localizes to nascent phagocytic cups during Fcγ receptor-mediated phagocytosis, where it displays the same spatial and temporal distribution as the actin cytoskeleton. Down-regulation of LSP1 severely reduces the phagocytic activity of macrophages, clearly demonstrating a crucial role for this protein in Fcγ receptor-mediated phagocytosis. We also find that LSP1 binds to the class I molecular motor myosin1e. LSP1 interacts with the SH3 domain of myosin1e, and the localization and dynamics of both proteins in nascent phagocytic cups mirror those of actin. Furthermore, inhibition of LSP1-myosin1e and LSP1-actin interactions profoundly impairs pseudopodial formation around opsonized targets and their subsequent internalization. Thus the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling during Fcγ receptor-driven phagocytosis.

  18. Unconventional myosins, actin dynamics and endocytosis: a ménage à trois?

    PubMed

    Soldati, Thierry

    2003-06-01

    Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation. PMID:12753645

  19. Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

    PubMed Central

    Chen, Tiane; Hubbard, Ann; Murtazina, Rakhilya; Price, Jennifer; Yang, Jianbo; Cha, Boyoung; Sarker, Rafiquel; Donowitz, Mark

    2014-01-01

    ABSTRACT The intestinal brush border Na+/H+ exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus. PMID:24928903

  20. Analysis of the interactions between Rab GTPases and class V myosins.

    PubMed

    Lindsay, Andrew J; Miserey-Lenkei, Stéphanie; Goud, Bruno

    2015-01-01

    Myosins are actin-based motor proteins that are involved in a wide variety of cellular processes such as membrane transport, muscle contraction, and cell division. Humans have over 40 myosins that can be placed into 18 classes, the malfunctioning of a number of which can lead to disease. There are three members of the human class V myosin family, myosins Va, Vb, and Vc. People lacking functional myosin Va suffer from a rare autosomal recessive disease called Griscelli's Syndrome type I (GS1) that is characterized by severe neurological defects and partial albinism. Mutations in the myosin Vb gene lead to an epithelial disorder called microvillus inclusion disease (MVID) that is often fatal in infants. The class V myosins have been implicated in the transport of diverse cargoes such as melanosomes in pigment cells, synaptic vesicles in neurons, RNA transcripts in a variety of cell types, and organelles such as the endoplasmic reticulum. The Rab GTPases play a critical role in recruiting class V myosins to their cargo. We recently published a study in which we used the yeast two-hybrid system to systematically test myosin Va for its ability to interact with each member of the human Rab GTPase family. We present here a detailed description of this yeast two-hybrid "living chip" assay. Furthermore, we present a protocol for validating positive interactions obtained from this screen by coimmunoprecipitation. PMID:25800833

  1. A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

    PubMed Central

    Dror, Amiel A.; Song, Lin; Ron, Uri; Tan, Joshua T.; Shitrit, Alina Starovolsky; Fuchs, Helmut; Hasson, Tama; Ben-Tal, Nir; Sweeney, H. Lee; de Angelis, Martin Hrabe; Steel, Karen P.; Avraham, Karen B.

    2008-01-01

    Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness. PMID:18833301

  2. 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

  3. Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes*

    PubMed Central

    Bultema, Jarred J.; Boyle, Judith A.; Malenke, Parker B.; Martin, Faye E.; Dell'Angelica, Esteban C.; Cheney, Richard E.; Di Pietro, Santiago M.

    2014-01-01

    Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion. PMID:25324551

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

    PubMed

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

    2016-10-01

    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

  5. Molecular motors: nature's nanomachines.

    PubMed

    Tyreman, M J A; Molloy, J E

    2003-12-01

    Molecular motors are protein-based machines that convert chemical potential energy into mechanical work. This paper aims to introduce the non-specialist reader to molecular motors, in particular, acto-myosin, the prototype system for motor protein studies. These motors produce their driving force from changes in chemical potential arising directly from chemical reactions and are responsible for muscle contraction and a variety of other cell motilities.

  6. Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

    PubMed

    Milograna, Sarah Ribeiro; Ribeiro, Márcia Regina; Baqui, Munira Muhammad Abdel; McNamara, John Campbell

    2014-12-01

    The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²⁺/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean

  7. Functional connectivity delineates distinct roles of the inferior frontal cortex and pre-supplementary motor area in stop signal inhibition

    PubMed Central

    Duann, Jeng-Ren; Ide, Jaime S.; Luo, Xi; Li, Chiang-shan Ray

    2009-01-01

    The neural basis of motor response inhibition has drawn considerable attention in recent imaging literature. Many studies have employed the go/no-go or stop signal task to examine the neural processes underlying motor response inhibition. In particular, showing greater activity during no-go (stop) as compared to go trials and during stop success as compared to stop error trials, the right inferior prefrontal cortex (IFC) has been suggested by numerous studies as the cortical area mediating response inhibition. Many of these same studies as well as others have also implicated the pre-supplementary motor area (preSMA) in this process, in accord with a function of the medial prefrontal cortex in goal-directed action. Here we employed connectivity analyses to delineate the roles of IFC and preSMA during stop signal inhibition. Specifically, we hypothesized that, as an integral part of the ventral attention system, the IFC responds to a stop signal and expedites the stop process in the preSMA, the primary site of motor response inhibition. This hypothesis predicted that preSMA and primary motor cortex would show functional interconnectivity via the basal ganglia circuitry to mediate response execution or inhibition, whereas the IFC would influence the basal ganglia circuitry via connectivity with preSMA. The results of Granger causality analyses in 57 participants confirmed this hypothesis. Furthermore, psychophysiological interaction showed that, as compared to stop errors, stop successes evoked greater effective connectivity between the IFC and preSMA, providing additional support for this hypothesis. These new findings provided evidence critically differentiating the roles of IFC and preSMA during stop signal inhibition and have important implications for our understanding of the component processes of inhibitory control. PMID:19675251

  8. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  9. Motor directional tuning across brain areas: directional resonance and the role of inhibition for directional accuracy.

    PubMed

    Mahan, Margaret Y; Georgopoulos, Apostolos P

    2013-01-01

    Motor directional tuning (Georgopoulos et al., 1982) has been found in every brain area in which it has been sought for during the past 30-odd years. It is typically broad, with widely distributed preferred directions and a population signal that predicts accurately the direction of an upcoming reaching movement or isometric force pulse (Georgopoulos et al., 1992). What is the basis for such ubiquitous directional tuning? How does the tuning come about? What are the implications of directional tuning for understanding the brain mechanisms of movement in space? This review addresses these questions in the light of accumulated knowledge in various sub-fields of neuroscience and motor behavior. It is argued (a) that direction in space encompasses many aspects, from vision to muscles, (b) that there is a directional congruence among the central representations of these distributed "directions" arising from rough but orderly topographic connectivities among brain areas, (c) that broad directional tuning is the result of broad excitation limited by recurrent and non-recurrent (i.e., direct) inhibition within the preferred direction loci in brain areas, and (d) that the width of the directional tuning curve, modulated by local inhibitory mechanisms, is a parameter that determines the accuracy of the directional command. PMID:23720612

  10. Structural Basis for Myosin V Discrimination Between Distinct Cargoes

    SciTech Connect

    Pashkova,N.; Jin, Y.; Ramaswamy, S.; Weisman, L.

    2006-01-01

    Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo-specific receptors. Here we report the crystal structure at 2.2 {angstrom} of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo-specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong-shaped cargo-binding domain, and moreover are offset by 180{sup o}. The fact that the cargo-binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle-specific myosin V receptors.

  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

    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.

  12. TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule*

    PubMed Central

    Balchand, Sai K.; Mann, Barbara J.; Titus, Janel; Ross, Jennifer L.; Wadsworth, Patricia

    2015-01-01

    The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays. PMID:26018074

  13. Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport.

    PubMed

    Ali, M Yusuf; Kennedy, Guy G; Safer, Daniel; Trybus, Kathleen M; Sweeney, H Lee; Warshaw, David M

    2011-08-23

    Myosin Va (myoV) and myosin VI (myoVI) are processive molecular motors that transport cargo in opposite directions on actin tracks. Because these motors may bind to the same cargo in vivo, we developed an in vitro "tug of war" to characterize the stepping dynamics of single quantum-dot-labeled myoV and myoVI motors linked to a common cargo. MyoV dominates its myoVI partner 79% of the time. Regardless of which motor wins, its stepping rate slows due to the resistive load of the losing motor (myoV, 2.1 pN; myoVI, 1.4 pN). Interestingly, the losing motor steps backward in synchrony with the winning motor. With ADP present, myoVI acts as an anchor to prevent myoV from stepping forward. This model system emphasizes the physical communication between opposing motors bound to a common cargo and highlights the potential for modulating this interaction by changes in the cell's ionic milieu.

  14. Phosphorylation and actin activation of brain myosin.

    PubMed Central

    Barylko, B; Sobieszek, A

    1983-01-01

    A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin. Images Fig. 1. Fig. 3. PMID:11894951

  15. Biased Brownian motion mechanism for processivity and directionality of single-headed myosin-VI.

    PubMed

    Iwaki, Mitsuhiro; Iwane, Atsuko Hikikoshi; Ikebe, Mitsuo; Yanagida, Toshio

    2008-01-01

    Conventional form to function as a vesicle transporter is not a 'single molecule' but a coordinated 'two molecules'. The coordinated two molecules make it complicated to reveal its mechanism. To overcome the difficulty, we adopted a single-headed myosin-VI as a model protein. Myosin-VI is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. However, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting doubt on its processivity. Using single molecule techniques, we show that green fluorescent protein (GFP)-fused single-headed myosin-VI does not move processively. However, when coupled to a 200 nm polystyrene bead (comparable to an intracellular vesicle in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40 nm) steps. Furthermore, we found that a single-headed myosin-VI-bead complex moved more processively in a high-viscous solution (40-fold higher than water) similar to cellular environment. Because diffusion of the bead is 60-fold slower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing the head's rebinding following detachment and supporting processive movement of the bead-monomer complex. This investigation will help us understand how molecular motors utilize Brownian motion in cells.

  16. Analysis of Organelle Targeting by DIL Domains of the Arabidopsis Myosin XI Family

    PubMed Central

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2011-01-01

    The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the “DIL” domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL–YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs. PMID:22645548

  17. Mechanochemical tuning of myosin-I by the N-terminal region.

    PubMed

    Greenberg, Michael J; Lin, Tianming; Shuman, Henry; Ostap, E Michael

    2015-06-30

    Myosins are molecular motors that generate force to power a wide array of motile cellular functions. Myosins have the inherent ability to change their ATPase kinetics and force-generating properties when they encounter mechanical loads; however, little is known about the structural elements in myosin responsible for force sensing. Recent structural and biophysical studies have shown that myosin-I isoforms, Myosin-Ib (Myo1b) and Myosin-Ic (Myo1c), have similar unloaded kinetics and sequences but substantially different responses to forces that resist their working strokes. Myo1b has the properties of a tension-sensing anchor, slowing its actin-detachment kinetics by two orders of magnitude with just 1 pN of resisting force, whereas Myo1c has the properties of a slow transporter, generating power without slowing under 1-pN loads that would stall Myo1b. To examine the structural elements that lead to differences in force sensing, we used single-molecule and ensemble kinetic techniques to show that the myosin-I N-terminal region (NTR) plays a critical role in tuning myosin-I mechanochemistry. We found that replacing the Myo1c NTR with the Myo1b NTR changes the identity of the primary force-sensitive transition of Myo1c, resulting in sensitivity to forces of <2 pN. Additionally, we found that the NTR plays an important role in stabilizing the post-power-stroke conformation. These results identify the NTR as an important structural element in myosin force sensing and suggest a mechanism for generating diversity of function among myosin isoforms. PMID:26056287

  18. Regulation and function of the fission yeast myosins.

    PubMed

    East, Daniel A; Mulvihill, Daniel P

    2011-05-01

    It is now quarter of a century since the actin cytoskeleton was first described in the fission yeast, Schizosaccharomyces pombe. Since then, a substantial body of research has been undertaken on this tractable model organism, extending our knowledge of the organisation and function of the actomyosin cytoskeleton in fission yeast and eukaryotes in general. Yeast represents one of the simplest eukaryotic model systems that has been characterised to date, and its genome encodes genes for homologues of the majority of actin regulators and actin-binding proteins found in metazoan cells. The ease with which diverse methodologies can be used, together with the small number of myosins, makes fission yeast an attractive model system for actomyosin research and provides the opportunity to fully understand the biochemical and functional characteristics of all myosins within a single cell type. In this Commentary, we examine the differences between the five S. pombe myosins, and focus on how these reflect the diversity of their functions. We go on to examine the role that the actin cytoskeleton plays in regulating the myosin motor activity and function, and finally explore how research in this simple unicellular organism is providing insights into the substantial impacts these motors can have on development and viability in multicellular higher-order eukaryotes. PMID:21502135

  19. The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V-Actin Bond

    PubMed Central

    Gebhardt, J. Christof M.; Ökten, Zeynep; Rief, Matthias

    2010-01-01

    Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense. PMID:20338849

  20. Evidence that Myosin Activity Opposes Microtubule-based Axonal Transport of Mitochondria

    PubMed Central

    Pathak, Divya; Sepp, Katharine J.; Hollenbeck, Peter J.

    2010-01-01

    Neurons transport and position mitochondria using a combination of saltatory, bidirectional movements and stationary docking. Axonal mitochondria move along microtubules (MTs) using kinesin and dynein motors, but actin and myosin also play a poorly-defined role in their traffic. To ascertain this role, we have used RNA interference to deplete specific myosin motors in cultured Drosophila neurons and quantified the effects on mitochondrial motility. We produced a fly strain expressing the C. elegans RNA transporter SID-1 in neurons to increase the efficacy of RNAi in primary cultures. These neurons exhibited significantly increased RNAi-mediated knockdown of gene expression compared to neurons not expressing this transporter. Using this system, we observed a significant increase in mitochondrial transport upon myosin V depletion. Mitochondrial mean velocity and duty cycle were augmented in both anterograde and retrograde directions, and the fraction of mitochondrial flux contained in long runs almost doubled for anterograde movement. Myosin VI depletion increased the same movement parameters, but was selective for retrograde movement, while myosin II depletion produced no phenotype. An additional effect of myosin V depletion was an increase in mitochondrial length. These data indicate that myosin V and VI play related but distinct roles in regulating MT-based mitochondrial movement: they oppose, rather than complement protracted MT-based movements and perhaps facilitate organelle docking. PMID:20592219

  1. Median raphe stimulation-induced motor inhibition concurrent with suppression of type 1 and type 2 hippocampal theta.

    PubMed

    Bland, Brian H; Bland, Cheryl E; MacIver, M Bruce

    2016-03-01

    This study investigated behavioral, anatomical and electrophysiological effects produced by electrical stimulation of posterior hypothalamic (PH) or median raphe (MR) nuclei, independently and during combined stimulation of both PH and MR. These three stimulation conditions were applied during spontaneous behavior in an open field and during PH stimulation-induced wheel running, while simultaneously recording hippocampal (HPC) field activity. An additional objective was to determine the effects of MR stimulation on Type 1 movement related theta and Type 2 sensory processing related theta. To achieve the latter, when behavioral studies were completed we studied the same rats under urethane anesthesia and then during urethane anesthesia with the addition of atropine sulfate (ATSO4). Here we demonstrated that electrical stimulation of a localized region of the MR nucleus resulted in a profound inhibition of both spontaneously occurring theta related motor behaviors and the theta related motor behaviors induced by electrical stimulation of the PH nucleus. Furthermore, this motor inhibition occurred concurrently with strong suppression of hippocampal theta field oscillations in the freely moving rat, a condition where the theta recorded is Type 2 sensory processing theta occurring coincidently with Type 1 movement related theta (Bland, 1986). Our results indicate that motor inhibition resulted from stimulation of neurons located in the mid central region of the MR, while stimulation in adjacent regions produced variable responses, including movements and theta activity. The present study provided evidence that the pharmacological basis of the suppression of Type 2 sensory processing HPC theta was cholinergic. However, MR inhibition of PH-induced wheel running was not affected by cholinergic blockade, which blocks Type 2 theta, indicating that MR stimulation-induced motor inhibition also requires the suppression of Type 1 theta.

  2. A Role for Myosin 1e in Cortical Granule Exocytosis in Xenopus Oocytes*s

    PubMed Central

    Schietroma, Cataldo; Yu, Hoi-Ying; Wagner, Mark C.; Umbach, Joy A.; Bement, William M.; Gundersen, Cameron B.

    2010-01-01

    Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin1e in cortical granule exocytosis. Myosin1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin1e relocates to the surface of cortical granules. Overexpression of myosin1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis. PMID:17702742

  3. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics.

    PubMed

    Chang, Ching-Wei; Kumar, Sanjay

    2015-09-04

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.

  4. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  5. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

    PubMed Central

    Harris, Samantha P.; Belknap, Betty; Van Sciver, Robert E.; White, Howard D.; Galkin, Vitold E.

    2016-01-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure–function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the “open” structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  6. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

  7. Inhibition of the pontine Kölliker-Fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat.

    PubMed

    Bautista, T G; Dutschmann, M

    2014-05-16

    The pontine Kölliker-Fuse nucleus (KF) has established functions in the regulation of inspiratory-expiratory phase transition and the regulation of upper airway patency via laryngeal valving mechanisms. Here we studied the role of the KF in the gating and modulation of eupneic hypoglossal motor activity (HNA) using the in situ perfused brainstem preparation, which displays robust inspiratory HNA. Microinjection of glutamate into the KF area triggered complex and often biphasic modulation (excitation/inhibition or inhibition/excitation) of HNA. Subsequent transient pharmacological inhibition of KF by unilateral microinjection of GABA-A receptor agonist isoguvacine reduced HNA and while bilateral microinjections completely abolished HNA. Our results indicate that mixed and overlapping KF pre-motor neurons provide eupneic drive for inspiratory HNA and postinspiratory vagal nerve activity. Both motor activities have important functions in the regulation of upper airway patency during eupnea but also during various oro-pharyngeal behaviors. These results have potential implications in the contribution of state-dependent modulation of KF hypoglossal pre-motor neurons during sleep-wake cycle to obstructive sleep apnea. PMID:24603053

  8. Inhibition of the pontine Kölliker-Fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat.

    PubMed

    Bautista, T G; Dutschmann, M

    2014-05-16

    The pontine Kölliker-Fuse nucleus (KF) has established functions in the regulation of inspiratory-expiratory phase transition and the regulation of upper airway patency via laryngeal valving mechanisms. Here we studied the role of the KF in the gating and modulation of eupneic hypoglossal motor activity (HNA) using the in situ perfused brainstem preparation, which displays robust inspiratory HNA. Microinjection of glutamate into the KF area triggered complex and often biphasic modulation (excitation/inhibition or inhibition/excitation) of HNA. Subsequent transient pharmacological inhibition of KF by unilateral microinjection of GABA-A receptor agonist isoguvacine reduced HNA and while bilateral microinjections completely abolished HNA. Our results indicate that mixed and overlapping KF pre-motor neurons provide eupneic drive for inspiratory HNA and postinspiratory vagal nerve activity. Both motor activities have important functions in the regulation of upper airway patency during eupnea but also during various oro-pharyngeal behaviors. These results have potential implications in the contribution of state-dependent modulation of KF hypoglossal pre-motor neurons during sleep-wake cycle to obstructive sleep apnea.

  9. Acetylcholinesterase inhibition and locomotor function after motor-sensory cortex impact injury.

    PubMed

    Holschneider, Daniel P; Guo, Yumei; Roch, Margareth; Norman, Keith M; Scremin, Oscar U

    2011-09-01

    Traumatic brain injury (TBI) induces transient or persistent dysfunction of gait and balance. Enhancement of cholinergic transmission has been reported to accelerate recovery of cognitive function after TBI, but the effects of this intervention on locomotor activity remain largely unexplored. The hypothesis that enhancement of cholinergic function by inhibition of acetylcholinesterase (AChE) improves locomotion following TBI was tested in Sprague-Dawley male rats after a unilateral controlled cortical impact (CCI) injury of the motor-sensory cortex. Locomotion was tested by time to fall on the constant speed and accelerating Rotarod, placement errors and time to cross while walking through a horizontal ladder, activity monitoring in the home cages, and rearing behavior. Assessments were performed the 1st and 2nd day and the 1st, 2nd, and 3rd week after TBI. The AChE inhibitor physostigmine hemisulfate (PHY) was administered continuously via osmotic minipumps implanted subcutaneously at the rates of 1.6-12.8 μmol/kg/day. All measures of locomotion were impaired by TBI and recovered to initial levels between 1 and 3 weeks post-TBI, with the exception of the maximum speed achievable on the accelerating Rotarod, as well as rearing in the open field. PHY improved performance in the accelerating Rotarod at 1.6 and 3.2 μmol/kg/day (AChE activity 95 and 78% of control, respectively), however, higher doses induced progressive deterioration. No effect or worsening of outcomes was observed at all PHY doses for home cage activity, rearing, and horizontal ladder walking. Potential benefits of cholinesterase inhibition on locomotor function have to be weighed against the evidence of the narrow range of useful doses. PMID:21787180

  10. Treadmill exercise ameliorates motor dysfunction through inhibition of Purkinje cell loss in cerebellum of valproic acid-induced autistic rats

    PubMed Central

    Cho, Han-Sam; Kim, Tae-Woon; Ji, Eun-Sang; Park, Hye-Sang; Shin, Mal-Soon; Baek, Seung-Soo

    2016-01-01

    Autism is a complex developmental disorder with impairments in social interaction, communication, repetitive behavior and motor skills. Exercise enhances cognitive function, ameliorates motor dysfunction, and provides protective profits against neurodegeneration. In the present study, we evaluated the effect of treadmill exercise on the motor coordination and Purkinje cell loss in relation with reactive astrocytes and microglial activation in the cerebellum using valproic acid (VPA)-induced autism rat model. On the 12th day of pregnancy, the pregnant rats in the VPA-exposed group received intraperitoneal injections of 600-mg/kg VPA. After birth, the rat pups were divided into four groups: the control group, the exercise group, the VPA-treated group, the VPA-treated and exercise group. The rat pups in the exercise groups were forced to run on a treadmill for 30 min once a day, 5 times a week for 4 weeks. In the present results, motor balance and coordination was disturbed by induction of autism, in contrast, treadmill exercise alleviated motor dysfunction in the autistic rats. Purkinje cell loss, reactive astrocytes, and microglial activation were occurred by induction of autism, in contrast, treadmill exercise enhanced survival rate of Purkinje neurons through inhibition of reactive astrocytes and microglia in the autistic rats. The present study showed that exercise may provide a potential therapeutic strategy for the alleviation of motor dysfunction in autistic patients. PMID:27656625

  11. Treadmill exercise ameliorates motor dysfunction through inhibition of Purkinje cell loss in cerebellum of valproic acid-induced autistic rats.

    PubMed

    Cho, Han-Sam; Kim, Tae-Woon; Ji, Eun-Sang; Park, Hye-Sang; Shin, Mal-Soon; Baek, Seung-Soo

    2016-08-01

    Autism is a complex developmental disorder with impairments in social interaction, communication, repetitive behavior and motor skills. Exercise enhances cognitive function, ameliorates motor dysfunction, and provides protective profits against neurodegeneration. In the present study, we evaluated the effect of treadmill exercise on the motor coordination and Purkinje cell loss in relation with reactive astrocytes and microglial activation in the cerebellum using valproic acid (VPA)-induced autism rat model. On the 12th day of pregnancy, the pregnant rats in the VPA-exposed group received intraperitoneal injections of 600-mg/kg VPA. After birth, the rat pups were divided into four groups: the control group, the exercise group, the VPA-treated group, the VPA-treated and exercise group. The rat pups in the exercise groups were forced to run on a treadmill for 30 min once a day, 5 times a week for 4 weeks. In the present results, motor balance and coordination was disturbed by induction of autism, in contrast, treadmill exercise alleviated motor dysfunction in the autistic rats. Purkinje cell loss, reactive astrocytes, and microglial activation were occurred by induction of autism, in contrast, treadmill exercise enhanced survival rate of Purkinje neurons through inhibition of reactive astrocytes and microglia in the autistic rats. The present study showed that exercise may provide a potential therapeutic strategy for the alleviation of motor dysfunction in autistic patients. PMID:27656625

  12. Treadmill exercise ameliorates motor dysfunction through inhibition of Purkinje cell loss in cerebellum of valproic acid-induced autistic rats

    PubMed Central

    Cho, Han-Sam; Kim, Tae-Woon; Ji, Eun-Sang; Park, Hye-Sang; Shin, Mal-Soon; Baek, Seung-Soo

    2016-01-01

    Autism is a complex developmental disorder with impairments in social interaction, communication, repetitive behavior and motor skills. Exercise enhances cognitive function, ameliorates motor dysfunction, and provides protective profits against neurodegeneration. In the present study, we evaluated the effect of treadmill exercise on the motor coordination and Purkinje cell loss in relation with reactive astrocytes and microglial activation in the cerebellum using valproic acid (VPA)-induced autism rat model. On the 12th day of pregnancy, the pregnant rats in the VPA-exposed group received intraperitoneal injections of 600-mg/kg VPA. After birth, the rat pups were divided into four groups: the control group, the exercise group, the VPA-treated group, the VPA-treated and exercise group. The rat pups in the exercise groups were forced to run on a treadmill for 30 min once a day, 5 times a week for 4 weeks. In the present results, motor balance and coordination was disturbed by induction of autism, in contrast, treadmill exercise alleviated motor dysfunction in the autistic rats. Purkinje cell loss, reactive astrocytes, and microglial activation were occurred by induction of autism, in contrast, treadmill exercise enhanced survival rate of Purkinje neurons through inhibition of reactive astrocytes and microglia in the autistic rats. The present study showed that exercise may provide a potential therapeutic strategy for the alleviation of motor dysfunction in autistic patients.

  13. Better Working Memory and Motor Inhibition in Children Who Delayed Gratification

    PubMed Central

    Yu, Junhong; Kam, Chi-Ming; Lee, Tatia M. C.

    2016-01-01

    Background: Despite the extensive research on delayed gratification over the past few decades, the neurocognitive processes that subserve delayed gratification remains unclear. As an exploratory step in studying these processes, the present study aims to describe the executive function profiles of children who were successful at delaying gratification and those who were not. Methods: A total of 138 kindergarten students (65 males, 73 females; Mage = 44 months, SD = 3.5; age range = 37–53 months) were administered a delayed gratification task, a 1-back test, a Day/night Stroop test and a Go/no-go test. The outcome measures of these tests were then analyzed between groups using a Multivariate Analysis of Variance, and subsequently a Multivariate Analysis of Covariance incorporating age as a covariate. Results: Children who were successful in delaying gratification were significantly older and had significantly better outcomes in the 1-back test and go/no-go test. With the exception of the number of hits in the go/no-go test, all other group differences remained significant after controlling for age. Conclusion: Children who were successful in delaying gratification showed better working memory and motor inhibition relative to those who failed the delayed gratification task. The implications of these findings are discussed. PMID:27493638

  14. Structure of the light chain-binding domain of myosin V

    PubMed Central

    Terrak, Mohammed; Rebowski, Grzegorz; Lu, Renne C.; Grabarek, Zenon; Dominguez, Roberto

    2005-01-01

    Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of ≈36 nm along the actin helical track, depend on its unusually long light chain-binding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2–IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM–IQ complexes, comparative sequence analysis, and the previously determined structures of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD of myosin V. The IQs of myosin V are distributed into three pairs. There appear to be specific cooperative interactions between light chains within each IQ pair, but little or no interaction between pairs, providing flexibility at their junctions. The second and third IQ pairs each present a light chain, whether CaM or a CaM-related molecule, bound in a noncanonical extended conformation in which the N-lobe does not interact with the IQ motif. The resulting free N-lobes may engage in protein–protein interactions. The extended conformation is characteristic of the single IQ of myosin VI and is common throughout the myosin superfamily. The model points to a prominent role of the LCBD in the function, regulation, and molecular interactions of myosin V. PMID:16120677

  15. Calcium can mobilize and activate myosin-VI

    PubMed Central

    Batters, Christopher; Brack, Dario; Ellrich, Heike; Averbeck, Beate; Veigel, Claudia

    2016-01-01

    The ability to coordinate the timing of motor protein activation lies at the center of a wide range of cellular motile processes including endocytosis, cell division, and cancer cell migration. We show that calcium dramatically alters the conformation and activity of the myosin-VI motor implicated in pivotal steps of these processes. We resolved the change in motor conformation and in structural flexibility using single particle analysis of electron microscopic data and identified interacting domains using fluorescence spectroscopy. We discovered that calcium binding to calmodulin increases the binding affinity by a factor of 2,500 for a bipartite binding site on myosin-VI. The ability of calcium-calmodulin to seek out and bridge between binding site components directs a major rearrangement of the motor from a compact dormant state into a cargo binding primed state that is nonmotile. The lack of motility at high calcium is due to calmodulin switching to a higher affinity binding site, which leaves the original IQ-motif exposed, thereby destabilizing the lever arm. The return to low calcium can either restabilize the lever arm, required for translocating the cargo-bound motors toward the center of the cell, or refold the cargo-free motors into an inactive state ready for the next cellular calcium flux. PMID:26811464

  16. Striatal D1- and D2-type dopamine receptors are linked to motor response inhibition in human subjects.

    PubMed

    Robertson, Chelsea L; Ishibashi, Kenji; Mandelkern, Mark A; Brown, Amira K; Ghahremani, Dara G; Sabb, Fred; Bilder, Robert; Cannon, Tyrone; Borg, Jacqueline; London, Edythe D

    2015-04-15

    Motor response inhibition is mediated by neural circuits involving dopaminergic transmission; however, the relative contributions of dopaminergic signaling via D1- and D2-type receptors are unclear. Although evidence supports dissociable contributions of D1- and D2-type receptors to response inhibition in rats and associations of D2-type receptors to response inhibition in humans, the relationship between D1-type receptors and response inhibition has not been evaluated in humans. Here, we tested whether individual differences in striatal D1- and D2-type receptors are related to response inhibition in human subjects, possibly in opposing ways. Thirty-one volunteers participated. Response inhibition was indexed by stop-signal reaction time on the stop-signal task and commission errors on the continuous performance task, and tested for association with striatal D1- and D2-type receptor availability [binding potential referred to nondisplaceable uptake (BPND)], measured using positron emission tomography with [(11)C]NNC-112 and [(18)F]fallypride, respectively. Stop-signal reaction time was negatively correlated with D1- and D2-type BPND in whole striatum, with significant relationships involving the dorsal striatum, but not the ventral striatum, and no significant correlations involving the continuous performance task. The results indicate that dopamine D1- and D2-type receptors are associated with response inhibition, and identify the dorsal striatum as an important locus of dopaminergic control in stopping. Moreover, the similar contribution of both receptor subtypes suggests the importance of a relative balance between phasic and tonic dopaminergic activity subserved by D1- and D2-type receptors, respectively, in support of response inhibition. The results also suggest that the stop-signal task and the continuous performance task use different neurochemical mechanisms subserving motor response inhibition. PMID:25878272

  17. Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

    PubMed

    Yao, Lin-Lin; Shen, Mei; Lu, Zekuan; Ikebe, Mitsuo; Li, Xiang-dong

    2016-04-01

    Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5. PMID:26912658

  18. Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin.

    PubMed

    Iwai, Sosuke; Chaen, Shigeru

    2007-05-25

    The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.

  19. Reconciling the influence of task-set switching and motor inhibition processes on stop signal after-effects.

    PubMed

    Anguera, Joaquin A; Lyman, Kyle; Zanto, Theodore P; Bollinger, Jacob; Gazzaley, Adam

    2013-01-01

    Executive response functions can be affected by preceding events, even if they are no longer associated with the current task at hand. For example, studies utilizing the stop signal task have reported slower response times to "GO" stimuli when the preceding trial involved the presentation of a "STOP" signal. However, the neural mechanisms that underlie this behavioral after-effect are unclear. To address this, behavioral and electroencephalography (EEG) measures were examined in 18 young adults (18-30 years) on "GO" trials following a previously "Successful Inhibition" trial (pSI), a previously "Failed Inhibition" trial (pFI), and a previous "GO" trial (pGO). Like previous research, slower response times were observed during both pSI and pFI trials (i.e., "GO" trials that were preceded by a successful and unsuccessful inhibition trial, respectively) compared to pGO trials (i.e., "GO" trials that were preceded by another "GO" trial). Interestingly, response time slowing was greater during pSI trials compared to pFI trials, suggesting executive control is influenced by both task set switching and persisting motor inhibition processes. Follow-up behavioral analyses indicated that these effects resulted from between-trial control adjustments rather than repetition priming effects. Analyses of inter-electrode coherence (IEC) and inter-trial coherence (ITC) indicated that both pSI and pFI trials showed greater phase synchrony during the inter-trial interval compared to pGO trials. Unlike the IEC findings, differential ITC was present within the beta and alpha frequency bands in line with the observed behavior (pSI > pFI > pGO), suggestive of more consistent phase synchrony involving motor inhibition processes during the ITI at a regional level. These findings suggest that between-trial control adjustments involved with task-set switching and motor inhibition processes influence subsequent performance, providing new insights into the dynamic nature of executive control.

  20. Identification, modeling, and characterization studies of Tetrahymena thermophila myosin FERM domains suggests a conserved core fold but functional differences.

    PubMed

    Martin, Che L; Singh, Shaneen M

    2015-11-01

    Myosins (MYO) define a superfamily of motor proteins which facilitate movement along cytoskeletal actin filaments in an ATP-dependent manner. To date, over 30 classes of myosin have been defined that vary in their roles and distribution across different taxa. The multidomain tail of myosin is responsible for the observed functional differences in different myosin classes facilitating differential binding to different cargos. One domain found in this region, the FERM domain, is found in several diverse proteins and is involved in many biological functions ranging from cell adhesion and actin-driven cytoskeleton assembly to cell signaling. Recently, new classes of unconventional myosin have been identified in Tetrahymena thermophila. In this study, we have identified, modeled, and characterized eight FERM domains from the unconventional T. thermophila myosins as their complete functional MyTH4-FERM cassettes. Our results reveal notable sequence, structural, and electrostatic differences between T. thermophila and other characterized FERM domains. Specifically, T. thermophila FERM domains contain helical inserts or extensions, which contribute to significant differences in surface electrostatic profiles of T. thermophila myosin FERMs when compared to the conventional FERM domains. Analyses of the modeled domains reveal differences in key functional residues as well as phosphoinositide-binding signatures and affinities. The work presented here broadens the scope of our understanding of myosin classes and their inherent functions, and provides a platform for experimentalists to design rational experimental studies to test the functional roles for T. thermophila myosins. PMID:26492945

  1. 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.

  2. Identification, modeling, and characterization studies of Tetrahymena thermophila myosin FERM domains suggests a conserved core fold but functional differences.

    PubMed

    Martin, Che L; Singh, Shaneen M

    2015-11-01

    Myosins (MYO) define a superfamily of motor proteins which facilitate movement along cytoskeletal actin filaments in an ATP-dependent manner. To date, over 30 classes of myosin have been defined that vary in their roles and distribution across different taxa. The multidomain tail of myosin is responsible for the observed functional differences in different myosin classes facilitating differential binding to different cargos. One domain found in this region, the FERM domain, is found in several diverse proteins and is involved in many biological functions ranging from cell adhesion and actin-driven cytoskeleton assembly to cell signaling. Recently, new classes of unconventional myosin have been identified in Tetrahymena thermophila. In this study, we have identified, modeled, and characterized eight FERM domains from the unconventional T. thermophila myosins as their complete functional MyTH4-FERM cassettes. Our results reveal notable sequence, structural, and electrostatic differences between T. thermophila and other characterized FERM domains. Specifically, T. thermophila FERM domains contain helical inserts or extensions, which contribute to significant differences in surface electrostatic profiles of T. thermophila myosin FERMs when compared to the conventional FERM domains. Analyses of the modeled domains reveal differences in key functional residues as well as phosphoinositide-binding signatures and affinities. The work presented here broadens the scope of our understanding of myosin classes and their inherent functions, and provides a platform for experimentalists to design rational experimental studies to test the functional roles for T. thermophila myosins.

  3. Myosin light chain genes in the turkey (Meleagris gallopavo).

    PubMed

    Chaves, L D; Ostroski, B J; Reed, K M

    2003-01-01

    Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.

  4. 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

  5. Naloxone reduces the amplitude of IPSPs evoked in lumbar motoneurons by reticular stimulation during carbachol-induced motor inhibition.

    PubMed

    Xi, M C; Liu, R H; Yamuy, J; Morales, F R; Chase, M H

    1999-02-20

    During active sleep or carbachol-induced motor inhibition, electrical stimulation of the medullary nucleus reticularis gigantocellularis (NRGc) evoked large amplitude, glycinergic inhibitory postsynaptic potentials (IPSPs) in cat motoneurons. The present study was directed to determine whether these IPSPs, that are specific to the state of active sleep, are modulated by opioid peptides. Accordingly, intracellular recordings were obtained from lumbar motoneurons of acute decerebrate cats during carbachol-induced motor inhibition while an opiate receptor antagonist, naloxone, was microiontophoretically released next to the recorded cells. Naloxone reversibly reduced by 26% the mean amplitude of NRGc-evoked IPSPs (1.9+/-0.2 mV (S.E.M.) vs. 1.4+/-0.2 mV; n=11, control and naloxone, respectively, p<0.05), but had no effect on the other waveform parameters of these IPSPs (e.g., latency-to-onset, latency-to-peak, duration, etc.). The mean resting membrane potential, input resistance and membrane time constant of motoneurons following naloxone ejection were not statistically different from those of the control. These data indicate that opioid peptides have a modulatory effect on NRGc-evoked IPSPs during carbachol-induced motor inhibition. We therefore suggest that endogenous opioid peptides may act as neuromodulators to regulate inhibitory glycinergic synaptic transmission at motoneurons during active sleep. PMID:10082872

  6. 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.

  7. Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

    NASA Astrophysics Data System (ADS)

    Nie, Wei

    The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

  8. Dynamics of myosin II organization into cortical contractile networks and fibers

    NASA Astrophysics Data System (ADS)

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel; Jedlicka, Sabrina; Vavylonis, Dimitrios

    2014-03-01

    The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

  9. Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium.

    PubMed

    Uyeda, T Q; Yumura, S

    2000-04-15

    The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological, and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis. PMID:10816252

  10. Metal cation controls myosin and actomyosin kinetics

    PubMed Central

    Tkachev, Yaroslav V; Ge, Jinghua; Negrashov, Igor V; Nesmelov, Yuri E

    2013-01-01

    We have perturbed myosin nucleotide binding site with magnesium-, manganese-, or calcium-nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time-resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation-specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number. PMID:24115140

  11. Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing.

    PubMed

    Brownstein, Zippora; Abu-Rayyan, Amal; Karfunkel-Doron, Daphne; Sirigu, Serena; Davidov, Bella; Shohat, Mordechai; Frydman, Moshe; Houdusse, Anne; Kanaan, Moien; Avraham, Karen B

    2014-06-01

    Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.

  12. Two-headed binding of a processive myosin to F-actin.

    PubMed

    Walker, M L; Burgess, S A; Sellers, J R; Wang, F; Hammer, J A; Trinick, J; Knight, P J

    2000-06-15

    Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation. PMID:10866203

  13. Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

    PubMed Central

    Giuliano, K A; Kolega, J; DeBiasio, R L; Taylor, D L

    1992-01-01

    The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors. Images PMID:1421576

  14. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI.

    PubMed

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-14

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  15. Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

    PubMed

    Rao, Mala V; Campbell, Jabbar; Palaniappan, Arti; Kumar, Asok; Nixon, Ralph A

    2016-04-01

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly, CAST

  16. Myosin: A Link between Streptococci and Heart

    NASA Astrophysics Data System (ADS)

    Krisher, Karen; Cunningham, Madeleine W.

    1985-01-01

    Murine monoclonal antibodies to Streptococcus pyogenes reacted with skeletal muscle myosin. High molecular weight proteins in extracts of human heart tissue that reacted with an antibody to S. pyogenes also reacted with a monoclonal antibody to ventricular myosin. Adsorption of the antibody to streptococci with S. pyogenes simultaneously removed reactivity of the antibody for either S. pyogenes or myosin. These results indicate that myosin shares immunodeterminants with a component of S. pyogenes.

  17. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

    PubMed Central

    Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

    2015-01-01

    The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

  18. 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.

  19. Development and initial assessment of a new paradigm for assessing cognitive and motor inhibition: the bimodal virtual-reality Stroop.

    PubMed

    Henry, Mylène; Joyal, Christian C; Nolin, Pierre

    2012-09-30

    Assessing and predicting inhibition in adults is a common assignment for clinicians. However, there is no single measure of inhibition that is complete, sensitive and enjoyable. The main goal of this study was to develop a virtual reality neuropsychological task (the bimodal VR-Stroop) capable of measuring both cognitive (control of internal and external interference) and motor inhibition (a go no-go paradigm with reaction time variation, commission errors and omissions). Preliminary data obtained with 71 healthy adult participants confirmed that the VR-Stroop is capable of eliciting the Stroop effect with bimodal stimuli. Initial validation data also suggested that measures of the VR-Stroop significantly correlate with measures of the Elevator counting with distracters, the Continuous Performance Task (CPT-II), and the Stop-it task. Finally, regression analyses indicated that commission errors and variability of reaction times at the VR-Stroop were significantly predicted by scores of the Elevator task and the CPT-II. These preliminary results suggest that the VR-Stroop is an interesting measure of cognitive and motor inhibition for adults, although confirmatory investigations are warranted.

  20. Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii.

    PubMed

    Kuznicki, J; Atkinson, M A; Korn, E D

    1984-07-25

    Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed. PMID:6235225

  1. Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

    PubMed Central

    Winkelmann, D. A.; Bourdieu, L.; Kinose, F.; Libchaber, A.

    1995-01-01

    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement. PMID:7787107

  2. Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

    PubMed

    Winkelmann, D A; Bourdieu, L; Kinose, F; Libchaber, A

    1995-04-01

    We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.

  3. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

    PubMed

    Spitznagel, Diana; O'Rourke, John F; Leddy, Neal; Hanrahan, Orla; Nolan, Derek P

    2010-01-01

    Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  4. Phase Dependency of the Human Primary Motor Cortex and Cholinergic Inhibition Cancelation During Beta tACS

    PubMed Central

    Guerra, Andrea; Pogosyan, Alek; Nowak, Magdalena; Tan, Huiling; Ferreri, Florinda; Di Lazzaro, Vincenzo; Brown, Peter

    2016-01-01

    The human motor cortex has a tendency to resonant activity at about 20 Hz so stimulation should more readily entrain neuronal populations at this frequency. We investigated whether and how different interneuronal circuits contribute to such resonance by using transcranial magnetic stimulation (TMS) during transcranial alternating current stimulation (tACS) at motor (20 Hz) and a nonmotor resonance frequency (7 Hz). We tested different TMS interneuronal protocols and triggered TMS pulses at different tACS phases. The effect of cholinergic short-latency afferent inhibition (SAI) was abolished by 20 Hz tACS, linking cortical beta activity to sensorimotor integration. However, this effect occurred regardless of the tACS phase. In contrast, 20 Hz tACS selectively modulated MEP size according to the phase of tACS during single pulse, GABAAergic short-interval intracortical inhibition (SICI) and glutamatergic intracortical facilitation (ICF). For SICI this phase effect was more marked during 20 Hz stimulation. Phase modulation of SICI also depended on whether or not spontaneous beta activity occurred at ~20 Hz, supporting an interaction effect between tACS and underlying circuit resonances. The present study provides in vivo evidence linking cortical beta activity to sensorimotor integration, and for beta oscillations in motor cortex being promoted by resonance in GABAAergic interneuronal circuits. PMID:27522077

  5. Contact-mediated inhibition between oligodendrocyte progenitor cells and motor exit point glia establishes the spinal cord transition zone.

    PubMed

    Smith, Cody J; Morris, Angela D; Welsh, Taylor G; Kucenas, Sarah

    2014-09-01

    Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.

  6. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a β-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity.

  7. Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty; Patel, Yashomati M.

    2008-10-15

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

  8. Novel Dictyostelium unconventional myosin, MyoM, has a putative RhoGEF domain.

    PubMed

    Oishi, N; Adachi, H; Sutoh, K

    2000-05-26

    We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM(-) mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis. PMID:10828443

  9. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

    PubMed

    Schönitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M

    2011-12-01

    Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

  10. Epithelial cell shape is regulated by Lulu proteins via myosin-II.

    PubMed

    Nakajima, Hiroyuki; Tanoue, Takuji

    2010-02-15

    Cell-shape change in epithelial structures is fundamental to animal morphogenesis. Recent studies identified myosin-II as the major generator of driving forces for cell-shape changes during morphogenesis. Lulu (Epb41l5) is a major regulator of morphogenesis, although the downstream molecular and cellular mechanisms remain obscure in mammals. In Drosophila and zebrafish, Lulu proteins were reported to negatively regulate Crumbs, an apical domain regulator, thus regulating morphogenesis. In this study, we show that mammalian Lulu activates myosin-II, thus regulating epithelial cell shape. In our experiments, Lulu expression in epithelial cells resulted in apical constriction and lateral elongation in the cells, accompanied by upregulation of myosin-II. The inhibition of myosin-II activity almost completely blocked this Lulu-driven cell-shape change. We further found that Rock participates in the myosin-II activation. Additionally, RNAi-mediated depletion of Lulu in epithelial cells resulted in disorganization of myosin-II and a concomitant loss of proper lateral domain organization in the cells. From these results, we propose that Lulu regulates epithelial cell shape by controlling myosin-II activity. PMID:20103536

  11. Redox-sensitive residue in the actin-binding interface of myosin.

    PubMed

    Moen, Rebecca J; Cornea, Sinziana; Oseid, Daniel E; Binder, Benjamin P; Klein, Jennifer C; Thomas, David D

    2014-10-24

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  12. Redox-sensitive residue in the actin-binding interface of myosin

    PubMed Central

    Moen, Rebecca J.; Cornea, Sinziana; Oseid, Daniel E.; Binder, Benjamin P.; Klein, Jennifer C.; Thomas, David D.

    2014-01-01

    We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress. PMID:25264102

  13. 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

  14. 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

  15. Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens.

    PubMed

    Yang, Long; Qin, Li; Liu, Guosheng; Peremyslov, Valera V; Dolja, Valerian V; Wei, Yangdou

    2014-09-23

    The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity. PMID:25201952

  16. A potentially exhaustive screening strategy reveals two novel divergent myosins in Dictyostelium.

    PubMed

    Schwarz, E C; Geissler, H; Soldati, T

    1999-01-01

    In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore, Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks. Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family in D. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection of myoK and myoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins. PMID:10403059

  17. Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

    PubMed Central

    Yang, Long; Qin, Li; Liu, Guosheng; Peremyslov, Valera V.; Dolja, Valerian V.; Wei, Yangdou

    2014-01-01

    The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity. PMID:25201952

  18. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

    PubMed Central

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia

    2014-01-01

    Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor’s oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite. PMID:24379364

  19. Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

    PubMed

    Taniguchi, Yukinori; Khatri, Bhavin S; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2010-07-01

    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal. PMID:20655854

  20. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis

    PubMed Central

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G.; González-Reyes, Acaimo; Martín-Bermudo, María D.

    2016-01-01

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies. PMID:26888436

  1. Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

    PubMed

    Valencia-Expósito, Andrea; Grosheva, Inna; Míguez, David G; González-Reyes, Acaimo; Martín-Bermudo, María D

    2016-02-18

    Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

  2. Myosin-I moves actin filaments on a phospholipid substrate: implications for membrane targeting

    PubMed Central

    1992-01-01

    Acanthamoeba myosin-I bound to substrates of nitrocellulose or planar lipid membranes on glass moved actin filaments at an average velocity of 0.2 micron/s. This movement required ATP and phosphorylation of the myosin-I heavy chain. We prepared planar lipid membranes on a glass support by passive fusion of lipid vesicles (Brian, A. A., and H. M. McConnell. 1984. Proc. Natl. Acad. Sci. USA. 81:6159-6163) composed of phosphatidylcholine and containing 0-40% phosphatidylserine. The mass of lipid that bound to the glass was the same for membranes of 2 and 20% phosphatidylserine in phosphatidylcholine and was sufficient to form a single bilayer. Myosin-I moved actin filaments on planar membranes of 5-40% but not 0-2% phosphatidylserine. At the low concentrations of phosphatidylserine, actin filaments tended to detach suggesting that less myosin-I was bound. We used the cooperative activation of Acanthamoeba myosin-I ATPase by low concentrations of actin to assess the association of phospholipids with myosin-I. Under conditions where activity depends on the binding of actin to the tail of myosin-I (Albanesi, J. P., H. Fujisaki, and E. D. Korn. 1985. J. Biol. Chem. 260:11174-11179), phospholipid vesicles with 5-40% phosphatidylserine inhibited ATPase activity. The motility and ATPase results demonstrate a specific interaction of the tail of myosin-I with physiological concentrations of phosphatidylserine. This interaction is sufficient to support motility and may provide a mechanism to target myosin-I to biological membranes. PMID:1530945

  3. A combined TMS-EEG study of short-latency afferent inhibition in the motor and dorsolateral prefrontal cortex.

    PubMed

    Noda, Yoshihiro; Cash, Robin F H; Zomorrodi, Reza; Dominguez, Luis Garcia; Farzan, Faranak; Rajji, Tarek K; Barr, Mera S; Chen, Robert; Daskalakis, Zafiris J; Blumberger, Daniel M

    2016-09-01

    Combined transcranial magnetic stimulation and electroencephalography (TMS-EEG) enables noninvasive neurophysiological investigation of the human cortex. A TMS paradigm of short-latency afferent inhibition (SAI) is characterized by attenuation of the motor-evoked potential (MEP) and modulation of N100 of the TMS-evoked potential (TEP) when TMS is delivered to motor cortex (M1) following median nerve stimulation. SAI is a marker of cholinergic activity in the motor cortex; however, the SAI has not been tested from the prefrontal cortex. We aimed to explore the effect of SAI in dorsolateral prefrontal cortex (DLPFC). SAI was examined in 12 healthy subjects with median nerve stimulation and TMS delivered to M1 and DLPFC at interstimulus intervals (ISIs) relative to the individual N20 latency. SAI in M1 was tested at the optimal ISI of N20 + 2 ms. SAI in DLPFC was investigated at a range of ISI from N20 + 2 to N20 + 20 ms to explore its temporal profile. For SAI in M1, the attenuation of MEP amplitude was correlated with an increase of TEP N100 from the left central area. A similar spatiotemporal neural signature of SAI in DLPFC was observed with a marked increase of N100 amplitude. SAI in DLPFC was maximal at ISI N20 + 4 ms at the left frontal area. These findings establish the neural signature of SAI in DLPFC. Future studies could explore whether DLPFC-SAI is neurophysiological marker of cholinergic dysfunction in cognitive disorders.

  4. Impaired Inhibition of Prepotent Motor Tendencies in Friedreich Ataxia Demonstrated by the Simon Interference Task

    ERIC Educational Resources Information Center

    Corben, L. A.; Akhlaghi, H.; Georgiou-Karistianis, N.; Bradshaw, J. L.; Egan, G. F.; Storey, E.; Churchyard, A. J.; Delatycki, M. B.

    2011-01-01

    Friedreich ataxia (FRDA) is the most common of the genetically inherited ataxias. We recently demonstrated that people with FRDA have impairment in motor planning--most likely because of pathology affecting the cerebral cortex and/or cerebello-cortical projections. We used the Simon interference task to examine how effective 13 individuals with…

  5. Regulation of the filament structure and assembly of Acanthamoeba myosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece.

    PubMed

    Liu, Xiong; Hong, Myoung-Soon; Shu, Shi; Yu, Shuhua; Korn, Edward D

    2013-01-01

    Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain. PMID:23248285

  6. Mapping interactions between myosin relay and converter domains that power muscle function.

    PubMed

    Kronert, William A; Melkani, Girish C; Melkani, Anju; Bernstein, Sanford I

    2014-05-01

    Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

  7. Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates

    PubMed Central

    Alcala, Diego B.; Haldeman, Brian D.; Brizendine, Richard K.; Krenc, Agata K.; Baker, Josh E.; Rock, Ronald S.; Cremo, Christine R.

    2016-01-01

    Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. PMID:27528075

  8. Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

    PubMed Central

    1993-01-01

    One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself. PMID

  9. Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation.

    PubMed

    Homburger, Julian R; Green, Eric M; Caleshu, Colleen; Sunitha, Margaret S; Taylor, Rebecca E; Ruppel, Kathleen M; Metpally, Raghu Prasad Rao; Colan, Steven D; Michels, Michelle; Day, Sharlene M; Olivotto, Iacopo; Bustamante, Carlos D; Dewey, Frederick E; Ho, Carolyn Y; Spudich, James A; Ashley, Euan A

    2016-06-14

    Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease. PMID:27247418

  10. Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation

    PubMed Central

    Homburger, Julian R.; Green, Eric M.; Caleshu, Colleen; Sunitha, Margaret S.; Taylor, Rebecca E.; Ruppel, Kathleen M.; Metpally, Raghu Prasad Rao; Colan, Steven D.; Michels, Michelle; Day, Sharlene M.; Olivotto, Iacopo; Bustamante, Carlos D.; Dewey, Frederick E.; Ho, Carolyn Y.; Spudich, James A.; Ashley, Euan A.

    2016-01-01

    Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease. PMID:27247418

  11. Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

    PubMed

    Haraguchi, Takeshi; Tominaga, Motoki; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2016-08-01

    Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator.

  12. Myosin XI-I is Mechanically and Enzymatically Unique Among Class-XI Myosins in Arabidopsis.

    PubMed

    Haraguchi, Takeshi; Tominaga, Motoki; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2016-08-01

    Arabidopsis possesses 13 genes encoding class-XI myosins. Among these, myosin XI-I is phylogenetically distant. To examine the molecular properties of Arabidopsis thaliana myosin XI-I (At myosin XI-I), we performed in vitro mechanical and enzymatic analyses using recombinant constructs of At myosin XI-I. Unlike other biochemically studied class-XI myosins, At myosin XI-I showed extremely low actin-activated ATPase activity (Vmax = 3.7 Pi s(-1) head(-1)). The actin-sliding velocity of At myosin XI-I was 0.25 µm s(-1), >10 times lower than those of other class-XI myosins. The ADP dissociation rate from acto-At myosin XI-I was 17 s(-1), accounting for the low actin-sliding velocity. In contrast, the apparent affinity for actin in the presence of ATP, estimated from Kapp (0.61 µM) of actin-activated ATPase, was extremely high. The equilibrium dissociation constant for actin was very low in both the presence and absence of ATP, indicating a high affinity for actin. To examine At myosin XI-I motility in vivo, green fluorescent protein-fused full-length At myosin XI-I was expressed in cultured Arabidopsis cells. At myosin XI-I localized not only on the nuclear envelope but also on small dots moving slowly (0.23 µm s(-1)) along actin filaments. Our results show that the properties of At myosin XI-I differ from those of other Arabidopsis class-XI myosins. The data suggest that At myosin XI-I does not function as a driving force for cytoplasmic streaming but regulates the organelle velocity, supports processive organelle movement or acts as a tension generator. PMID:27273580

  13. Binding of chara Myosin globular tail domain to phospholipid vesicles.

    PubMed

    Nunokawa, Shun-Ya; Anan, Hiromi; Shimada, Kiyo; Hachikubo, You; Kashiyama, Taku; Ito, Kohji; Yamamoto, Keiichi

    2007-11-01

    Binding of Chara myosin globular tail domain to phospholipid vesicles was investigated quantitatively. It was found that the globular tail domain binds to vesicles made from acidic phospholipids but not to those made from neutral phospholipids. This binding was weakened at high KCl concentration, suggesting that the binding is electrostatic by nature. The dissociation constant for the binding of the globular tail domain to 20% phosphatidylserine vesicles (similar to endoplasmic reticulum in acidic phospholipid contents) at 150 mM KCl was 273 nM. The free energy change due to this binding calculated from the dissociation constant was -37.3 kJ mol(-1). Thus the bond between the globular tail domain and membrane phospholipids would not be broken when the motor domain of Chara myosin moves along the actin filament using the energy of ATP hydrolysis (DeltaG degrees ' = -30.5 kJ mol(-1)). Our results suggested that direct binding of Chara myosin to the endoplasmic reticulum membrane through the globular tail domain could work satisfactorily in Chara cytoplasmic streaming. We also suggest a possible regulatory mechanism of cytoplasmic streaming including phosphorylation-dependent dissociation of the globular tail domain from the endoplasmic reticulum membrane.

  14. Reconciling the influence of task-set switching and motor inhibition processes on stop signal after-effects

    PubMed Central

    Anguera, Joaquin A.; Lyman, Kyle; Zanto, Theodore P.; Bollinger, Jacob; Gazzaley, Adam

    2013-01-01

    Executive response functions can be affected by preceding events, even if they are no longer associated with the current task at hand. For example, studies utilizing the stop signal task have reported slower response times to “GO” stimuli when the preceding trial involved the presentation of a “STOP” signal. However, the neural mechanisms that underlie this behavioral after-effect are unclear. To address this, behavioral and electroencephalography (EEG) measures were examined in 18 young adults (18–30 years) on “GO” trials following a previously “Successful Inhibition” trial (pSI), a previously “Failed Inhibition” trial (pFI), and a previous “GO” trial (pGO). Like previous research, slower response times were observed during both pSI and pFI trials (i.e., “GO” trials that were preceded by a successful and unsuccessful inhibition trial, respectively) compared to pGO trials (i.e., “GO” trials that were preceded by another “GO” trial). Interestingly, response time slowing was greater during pSI trials compared to pFI trials, suggesting executive control is influenced by both task set switching and persisting motor inhibition processes. Follow-up behavioral analyses indicated that these effects resulted from between-trial control adjustments rather than repetition priming effects. Analyses of inter-electrode coherence (IEC) and inter-trial coherence (ITC) indicated that both pSI and pFI trials showed greater phase synchrony during the inter-trial interval compared to pGO trials. Unlike the IEC findings, differential ITC was present within the beta and alpha frequency bands in line with the observed behavior (pSI > pFI > pGO), suggestive of more consistent phase synchrony involving motor inhibition processes during the ITI at a regional level. These findings suggest that between-trial control adjustments involved with task-set switching and motor inhibition processes influence subsequent performance, providing new insights into the

  15. Insights from the supplementary motor area syndrome in balancing movement initiation and inhibition

    PubMed Central

    Potgieser, A. R. E.; de Jong, B. M.; Wagemakers, M.; Hoving, E. W.; Groen, R. J. M.

    2014-01-01

    The supplementary motor area (SMA) syndrome is a characteristic neurosurgical syndrome that can occur after unilateral resection of the SMA. Clinical symptoms may vary from none to a global akinesia, predominantly on the contralateral side, with preserved muscle strength and mutism. A remarkable feature is that these symptoms completely resolve within weeks to months, leaving only a disturbance in alternating bimanual movements. In this review we give an overview of the old and new insights from the SMA syndrome and extrapolate these findings to seemingly unrelated diseases and symptoms such as Parkinson’s disease (PD) and tics. Furthermore, we integrate findings from lesion, stimulation and functional imaging studies to provide insight in the motor function of the SMA. PMID:25506324

  16. Flagellar localization of a novel isoform of myosin, myosin XXI, in Leishmania.

    PubMed

    Katta, Santharam S; Sahasrabuddhe, Amogh A; Gupta, Chhitar M

    2009-04-01

    Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids. PMID:19121339

  17. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  18. Motor Inhibition Test: Technical Report 13. Disadvantaged Children and Their First School Experiences. ETS-Head Start Longitudinal Study. Technical Report Series.

    ERIC Educational Resources Information Center

    Ward, William C.

    The Motor Inhibition Test, which measures "impulse control," requires the child to perform three motor acts. These acts are: walking a distance of six feet on a five-ince wide runway; drawing a line, using ruler and pencil, between two points 8-1/2 inches apart; and winding a toy jeep up to the rear of a toy two truck, a distance of 30 inches, so…

  19. Identification of T. gondii Myosin Light Chain-1 as a Direct Target of TachypleginA-2, a Small-Molecule Inhibitor of Parasite Motility and Invasion

    PubMed Central

    Leung, Jacqueline M.; Tran, Fanny; Pathak, Ravindra B.; Poupart, Séverine; Heaslip, Aoife T.; Ballif, Bryan A.; Westwood, Nicholas J.; Ward, Gary E.

    2014-01-01

    Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite’s life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by “click” chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite’s myosin motor. PMID:24892871

  20. Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

    PubMed

    Hayashida, Michikata; Tanifuji, Shota; Ma, Huan; Murakami, Noriko; Mochida, Sumiko

    2015-06-10

    Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns. PMID:26063922

  1. Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization

    PubMed Central

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel H.; Jedlicka, Sabrina S.; Vavylonis, Dimitrios

    2015-01-01

    The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. PMID:25641802

  2. Structure and interactions of myosin-binding protein C domain C0: cardiac-specific regulation of myosin at its neck?

    PubMed

    Ratti, Joyce; Rostkova, Elena; Gautel, Mathias; Pfuhl, Mark

    2011-04-01

    Myosin-binding protein C (MyBP-C) is a multidomain protein present in the thick filaments of striated muscles and is involved in both sarcomere formation and contraction regulation. The latter function is believed to be located at the N terminus, which is close to the motor domain of myosin. The cardiac isoform of MyBP-C is linked to hypertrophic cardiomyopathy. Here, we use NMR spectroscopy and biophysical and biochemical assays to study the three-dimensional structure and interactions of the cardiac-specific Ig-like domain C0, a part of cardiac MyBP-C of which little is known. The structure confirmed that C0 is a member of the IgI class of proteins, showing many of the characteristic features of this fold. Moreover, we identify a novel interaction between C0 and the regulatory light chain of myosin, thus placing the N terminus of the protein in proximity to the motor domain of myosin. This novel interaction is disrupted by several cardiomyopathy-linked mutations in the MYBPC3 gene. These results provide new insights into how cardiac MyBP-C incorporates in the sarcomere and how it can contribute to the regulation of muscle contraction.

  3. Electrophysiological properties of lumbar motoneurons in the alpha-chloralose-anesthetized cat during carbachol-induced motor inhibition.

    PubMed

    Xi, M C; Liu, R H; Yamuy, J; Morales, F R; Chase, M H

    1997-07-01

    The present study was undertaken 1) to examine the neuronal mechanisms responsible for the inhibition of spinal cord motoneurons that occurs in alpha-chloralose-anesthetized cats following the microinjection of carbachol into the nucleus pontis oralis (NPO), and 2) to determine whether the inhibitory mechanisms are the same as those that are responsible for the postsynaptic inhibition of motoneurons that is present during naturally occurring active sleep. Accordingly, the basic electrophysiological properties of lumbar motoneurons were examined, with the use of intracellular recording techniques, in cats anesthetized with alpha-chloralose and compared with those present during naturally occurring active sleep. The intrapontine administration of carbachol resulted in a sustained reduction in the amplitude of the spinal cord Ia monosynaptic reflex. Discrete large-amplitude inhibitory postsynaptic potentials (IPSPs), which are only present during the state of active sleep in the chronic cat, were also observed in high-gain recordings from lumbar motoneurons after the injection of carbachol. During carbachol-induced motor inhibition, lumbar motoneurons exhibited a statistically significant decrease in input resistance, membrane time constant and a reduction in the amplitude of the action potential's afterhyperpolarization. In addition, there was a statistically significant increase in rheobase and in the delay between the initial-segment (IS) and somadendritic (SD) portions of the action potential (IS-SD delay). There was a significant increase in the mean motoneuron resting membrane potential (i.e., hyperpolarization). The preceding changes in the electrophysiological properties of motoneurons, as well as the development of discrete IPSPs, indicate that lumbar motoneurons are postsynaptically inhibited after the intrapontine administration of carbachol in cats that are anesthetized with alpha-chloralose. These changes in the electrophysiological properties of lumbar

  4. Structure of the Single-lobe Myosin Light Chain C in Complex with the Light Chain-binding Domains of Myosin-1C Provides Insights into Divergent IQ Motif Recognition.

    PubMed

    Langelaan, David N; Liburd, Janine; Yang, Yidai; Miller, Emily; Chitayat, Seth; Crawley, Scott W; Côté, Graham P; Smith, Steven P

    2016-09-01

    Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC·myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N- and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.

  5. Engineering Circular Gliding of Actin Filaments Along Myosin-Patterned DNA Nanotube Rings To Study Long-Term Actin-Myosin Behaviors.

    PubMed

    Hariadi, Rizal F; Appukutty, Abhinav J; Sivaramakrishnan, Sivaraj

    2016-09-27

    Nature has evolved molecular motors that are critical in cellular processes occurring over broad time scales, ranging from seconds to years. Despite the importance of the long-term behavior of molecular machines, topics such as enzymatic lifetime are underexplored due to the lack of a suitable approach for monitoring motor activity over long time periods. Here, we developed an "O"-shaped Myosin Empowered Gliding Assay (OMEGA) that utilizes engineered micron-scale DNA nanotube rings with precise arrangements of myosin VI to trap gliding actin filaments. This circular gliding assay platform allows the same individual actin filament to glide over the same myosin ensemble (50-1000 motors per ring) multiple times. First, we systematically characterized the formation of DNA nanotubes rings with 4, 6, 8, and 10 helix circumferences. Individual actin filaments glide along the nanotube rings with high processivity for up to 12.8 revolutions or 11 min in run time. We then show actin gliding speed is robust to variation in motor number and independent of ring curvature within our sample space (ring diameter of 0.5-4 μm). As a model application of OMEGA, we then analyze motor-based mechanical influence on "stop-and-go" gliding behavior of actin filaments, revealing that the stop-to-go transition probability is dependent on motor flexibility. Our circular gliding assay may provide a closed-loop platform for monitoring long-term behavior of broad classes of molecular motors and enable characterization of motor robustness and long time scale nanomechanical processes.

  6. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins.

    PubMed

    Manstein, Dietmar J; Mulvihill, Daniel P

    2016-08-01

    The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic β- or γ-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures. PMID:27060364

  7. X-ray diffraction from flight muscle with a headless myosin mutation: implications for interpreting reflection patterns

    PubMed Central

    Iwamoto, Hiroyuki; Trombitás, Károly; Yagi, Naoto; Suggs, Jennifer A.; Bernstein, Sanford I.

    2014-01-01

    Fruit fly (Drosophila melanogaster) is one of the most useful animal models to study the causes and effects of hereditary diseases because of its rich genetic resources. It is especially suitable for studying myopathies caused by myosin mutations, because specific mutations can be induced to the flight muscle-specific myosin isoform, while leaving other isoforms intact. Here we describe an X-ray-diffraction-based method to evaluate the structural effects of mutations in contractile proteins in Drosophila indirect flight muscle. Specifically, we describe the effect of the headless myosin mutation, Mhc10-Y97, in which the motor domain of the myosin head is deleted, on the X-ray diffraction pattern. The loss of general integrity of the filament lattice is evident from the pattern. A striking observation, however, is the prominent meridional reflection at d = 14.5 nm, a hallmark for the regularity of the myosin-containing thick filament. This reflection has long been considered to arise mainly from the myosin head, but taking the 6th actin layer line reflection as an internal control, the 14.5-nm reflection is even stronger than that of wild-type muscle. We confirmed these results via electron microscopy, wherein image analysis revealed structures with a similar periodicity. These observations have major implications on the interpretation of myosin-based reflections. PMID:25400584

  8. Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability.

    PubMed

    Lee, Chi F; Melkani, Girish C; Yu, Qin; Suggs, Jennifer A; Kronert, William A; Suzuki, Yoko; Hipolito, Lori; Price, Maureen G; Epstein, Henry F; Bernstein, Sanford I

    2011-03-01

    UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick-thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

  9. Insight into the mechanism of fast movement of myosin from Chara corallina.

    PubMed

    Sumiyoshi, Hiroki; Ooguchi, Masami; Ooi, Atsushi; Okagaki, Tsuyoshi; Higashi-Fujime, Sugie

    2007-02-01

    Chara myosin, two-headed plant myosin belonging to class XI, slides F-actin at maximally 60 microm s(-1). To elucidate the mechanism of this fast sliding, we extensively investigated its mechanochemical properties. The maximum actin activated ATPase activity, Vmax, was 21.3 s(-1) head(-1) in a solution, but when myosin was immobilized on the surface, its activity was 57.6 s(-1) head(-1) at 2 mg ml(-1) of F-actin. The sliding velocity and the actin activated ATPase activity were greatly inhibited by ADP, suggesting that ADP dissociation was the rate limiting step. With the extensive assay of motility by varying the surface density, the duty ratio of Chara myosin was found to be 0.49-0.44 from velocity measurements and 0.34 from the landing rate analysis. At the surface density of 10 molecules microm(-2), Chara myosin exhibited pivot movement under physiological conditions. Based on the results obtained, we will discuss the sliding mechanism of Chara myosin according to the working stroke model in terms of its physiological aspects. aspects.

  10. The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II.

    PubMed

    Bloemink, Marieke J; Melkani, Girish C; Bernstein, Sanford I; Geeves, Michael A

    2016-01-22

    The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25-30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.

  11. 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.

  12. Size and speed of the working stroke of cardiac myosin in situ.

    PubMed

    Caremani, Marco; Pinzauti, Francesca; Reconditi, Massimo; Piazzesi, Gabriella; Stienen, Ger J M; Lombardi, Vincenzo; Linari, Marco

    2016-03-29

    The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 μm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach. PMID:26984499

  13. Size and speed of the working stroke of cardiac myosin in situ

    PubMed Central

    Caremani, Marco; Pinzauti, Francesca; Reconditi, Massimo; Piazzesi, Gabriella; Stienen, Ger J. M.; Lombardi, Vincenzo; Linari, Marco

    2016-01-01

    The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T (0.8–0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s−1 at high load to ∼8 nm⋅hs−1 and 6,000 s−1 at low load. Increases in sarcomere length (1.9–2.2 μm) and external [Ca2+]o (1–2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca2+-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach. PMID:26984499

  14. Response Inhibition in Motor and Oculomotor Conflict Tasks: Different Mechanisms, Different Dynamics?

    ERIC Educational Resources Information Center

    Wijnen, Jasper G.; Ridderinkhof, K. Richard

    2007-01-01

    Previous research has shown that the appearance of task-irrelevant abrupt onsets influences saccadic eye movements during visual search and may slow down manual reactions to target stimuli. Analysis of reaction time distributions in the present study offers evidence suggesting that top-down inhibition processes actively suppress oculomotor or…

  15. Conformational changes of myosin by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Lee, Ju.-Woon; Yook, Hong.-Sun; Lee, Kyong.-Haeng; Kim, Jae.-Hun; Kim, Woo.-Jung; Byun, Myung.-Woo

    2000-05-01

    Conformational and decompositional changes of bovine skeletal muscle myosin caused by gamma irradiation were studied for understanding the effects of irradiation treatment on myofibrillar proteins. Myosin solution and beef cuts were irradiated 0, 1, 3, 5 and 10 kGy. Competitive indirect enzyme linked immunosorbent assay (Ci-ELISA) showed that subunits of myosin were structurally modified with different patterns. Binding abilities of anti-myosin whole molecule and anti heavy meromyosin S-1 IgG, which were produced from rabbits, with irradiated myosin decreased in the same tendency depending upon the dose. Anti-light meromyosin IgG appeared to have the highest binding ability at 3 kGy. Irradiated beef cuts (≥5 kGy) could be identified by Ci-ELISA. Myosin solution became increasingly turbid with increasing dose. Hydrophobicity of myosin solution also increased by irradiation. Electrophoretic patterns showed that the myosin heavy chain disappeared and new bands were generated at higher molecular weight ranges.

  16. Force generation by skeletal muscle is controlled by mechanosensing in myosin filaments.

    PubMed

    Linari, Marco; Brunello, Elisabetta; Reconditi, Massimo; Fusi, Luca; Caremani, Marco; Narayanan, Theyencheri; Piazzesi, Gabriella; Lombardi, Vincenzo; Irving, Malcolm

    2015-12-10

    Contraction of both skeletal muscle and the heart is thought to be controlled by a calcium-dependent structural change in the actin-containing thin filaments, which permits the binding of myosin motors from the neighbouring thick filaments to drive filament sliding. Here we show by synchrotron small-angle X-ray diffraction of frog (Rana temporaria) single skeletal muscle cells that, although the well-known thin-filament mechanism is sufficient for regulation of muscle shortening against low load, force generation against high load requires a second permissive step linked to a change in the structure of the thick filament. The resting (switched 'OFF') structure of the thick filament is characterized by helical tracks of myosin motors on the filament surface and a short backbone periodicity. This OFF structure is almost completely preserved during low-load shortening, which is driven by a small fraction of constitutively active (switched 'ON') myosin motors outside thick-filament control. At higher load, these motors generate sufficient thick-filament stress to trigger the transition to its long-periodicity ON structure, unlocking the major population of motors required for high-load contraction. This concept of the thick filament as a regulatory mechanosensor provides a novel explanation for the dynamic and energetic properties of skeletal muscle. A similar mechanism probably operates in the heart. PMID:26560032

  17. Motoneuron properties during motor inhibition produced by microinjection of carbachol into the pontine reticular formation of the decerebrate cat.

    PubMed

    Morales, F R; Engelhardt, J K; Soja, P J; Pereda, A E; Chase, M H

    1987-04-01

    It is well established that cholinergic agonists, when injected into the pontine reticular formation in cats, produce a generalized suppression of motor activity (1, 3, 6, 14, 18, 27, 33, 50). The responsible neuronal mechanisms were explored by measuring ventral root activity, the amplitude of the Ia-monosynaptic reflex, and the basic electrophysiological properties of hindlimb motoneurons before and after carbachol was microinjected into the pontine reticular formation of decerebrate cats. Intrapontine microinjections of carbachol (0.25-1.0 microliter, 16 mg/ml) resulted in the tonic suppression of ventral root activity and a decrease in the amplitude of the Ia-monosynaptic reflex. An analysis of intracellular records from lumbar motoneurons during the suppression of motor activity induced by carbachol revealed a considerable decrease in input resistance and membrane time constant as well as a reduction in motoneuron excitability, as evidenced by a nearly twofold increase in rheobase. Discrete inhibitory postsynaptic potentials were also observed following carbachol administration. The changes in motoneuron properties (rheobase, input resistance, and membrane time constant), as well as the development of discrete inhibitory postsynaptic potentials, indicate that spinal cord motoneurons were postsynaptically inhibited following the pontine administration of carbachol. In addition, the inhibitory processes that arose after carbachol administration in the decerebrate cat were remarkably similar to those that are present during active sleep in the chronic cat. These findings suggest that the microinjection of carbachol into the pontine reticular formation activates the same brain stem-spinal cord system that is responsible for the postsynaptic inhibition of alpha-motoneurons that occurs during active sleep. PMID:3585456

  18. Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum.

    PubMed

    Katta, Santharam S; Tammana, Trinadh V Satish; Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2010-06-15

    Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum. PMID:20501700

  19. Effect of nucleotides on the orientation and mobility of myosin subfragment-1 in ghost muscle fiber.

    PubMed

    Pronina, O E; Wrzosek, A; Dabrowska, R; Borovikov, Yu S

    2005-10-01

    Using polarization fluorimetry, the orientation and mobility of 1,5-IAEDANS specifically bound to Cys707 of myosin subfragment-1 (S1) were studied in ghost muscle tropomyosin-containing fibers in the absence and in the presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes in the structural state of the myosin head during the ATPase cycle. Maximal differences in the probe orientation by 4 degrees and its mobility by 30% were found between actomyosin states in the presence of MgADP and MgATP. It is suggested that interaction of S1 with F-actin induces nucleotide-dependent rotation of the whole motor domain of the myosin head or only the dye-binding site and also change in the head mobility.

  20. Polymer Nanocomposites as a Facile Method for Engineering Acto-Myosin Networks at the Interface

    NASA Astrophysics Data System (ADS)

    Caporizzo, Matthew; Sun, Yujie; Goldman, Yale; Composto, Russell; Nano-Bio Interface Center Collaboration

    2011-03-01

    Filamentous actin acts as the rails for the molecular motor myosin in muscle contraction and intercellular mass transport. Consequently, understanding the process by which actin organizes, polymerizes, and binds is fundamental for the design of myosin based actuators capable of responding to external stimuli. Starting with atomically smooth, freshly cleaved mica optically coupled to glass slides, a random copolymer nanoparticle composite is engineered for in situ single molecule TIRF/AFM studies with controlled roughness, electrostatic binding strength, and binding site density. Four distinct regimes of actin binding are observed; no attachment, end-on attachment, weak side-on attachment, and side-on immobilization. Transitions between regimes are likely to mark competition between the affinity to charged nanoparticles and the inherent resistance of the semi-rigid filaments to bending. Surface conditions optimal for actin immobilization are identified, and Myosin V stepping kinetics are studied on the artificially immobilized filaments, confirming filament support of motility. Supported by NSF grant DMR-0425780.

  1. Structural kinetics of myosin by transient time-resolved FRET

    PubMed Central

    Nesmelov, Yuri E.; Agafonov, Roman V.; Negrashov, Igor V.; Blakely, Sarah E.; Titus, Margaret A.; Thomas, David D.

    2011-01-01

    For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)2FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)2FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism. PMID:21245357

  2. Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

    PubMed

    Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena; Sirkia, Helena; Clause, Jeffrey; Sourigues, Yannick; Johnsrud, Daniel O; Amigues, Beatrice; Cecchini, Marco; Gilbert, Susan P; Houdusse, Anne; Titus, Margaret A

    2016-05-24

    Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution. PMID:27166421

  3. Molecular dynamics simulation for the reversed power stroke motion of a myosin subfragment-1.

    PubMed

    Masuda, Tadashi

    2015-06-01

    Myosins are typical molecular motor proteins that convert the chemical energy from the ATP hydrolysis into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results already obtained, Masuda has proposed a hypothesis called the "Driven by Detachment" theory for the working principle of the myosins. This theory insists that the energy used during the power stroke of the myosins does not directly originate from the chemical energy of ATP, but is converted from the elastic energy within the molecule at the joint between the head and neck domains. One method for demonstrating the validity of this theory is a computational simulation using the molecular dynamics (MD) method. The MD software used was GROMACS. The target of the MD simulations was myosin subfragment-1 (S1), for which the initial structure was obtained from the Protein Data Bank entry 1M8Q. The AFM pull code of GROMACS was used to apply an external force of 17 pN at the end of the neck domain in the direction opposite to the power stroke to observe whether the myosin S1 takes the pre-power stroke conformation. The residues assumed to be engaged in the docking with an actin filament were fixed to the space. Starting from exactly the same initial position, 10 simulations were repeated by varying the random seeds for generating the initial velocities of the atoms. After 64ns of calculations, the myosin S1 took the conformation of the pre-power stroke state in which the neck domain was bent around the joint between the head and the neck domains. This result agrees with the prediction expected by the DbD theory, the validity of which may be established by conducting similar simulations for the other steps of the myosin working processes.

  4. Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron

    PubMed Central

    Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

    2016-01-01

    Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons. PMID:27570482

  5. Visualization of the mechanochemical coupling in myosin V using deac-aminoATP

    NASA Astrophysics Data System (ADS)

    Sakaoto, Takeshi; Webb, Martin; Forgacs, Eva; White, Howard; Sellers, Jim

    2010-03-01

    MyosinV is a twoheaded motor, which moves processively along an actin with ADP-release as the rate limiting step. The kinetic cycles of the two heads are gated by the internal strain each places on the other Mechanical studies suggest that there is tight coupling (i.e., one ATP is hydrolyzed per power stroke). We investigated the coordination between the ATPase mechanism of the two heads-myosin Va and directly visualized the binding and dissociation of nucleotide molecules, while simultaneously observing the stepping motion of the myosin V as it moved along an actin filament. To do this, we used an fluorescent labeled ATP analog, deac-aminoATP, which shows a 20fold increase in fluorescent intensity when bound to the active site of myosinV I directly demonstrate tight coupling between myosin V movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision. Supported by K99/R00 NIH grant.

  6. 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.

  7. 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.

  8. Transient inhibition of primary motor cortex suppresses hand muscle responses during a reactive reach to grasp.

    PubMed

    Bolton, David A E; Patel, Rupesh; Staines, W Richard; McIlroy, William E

    2011-10-24

    Rapid balance reactions such as compensatory reach to grasp represent important response strategies following unexpected loss of balance. While it has been assumed that early corrective actions arise from subcortical networks, recent research has prompted speculation about the potential role of cortical involvement. With reach to grasp reactions there is evidence of parallels in the control of perturbation-evoked reaching versus rapid voluntary reaching. However, the potential role of cortical involvement in such rapid balance reactions remains speculative. To test if cortical motor regions are involved we used continuous theta burst stimulation (cTBS) to temporarily suppress the hand area of primary motor cortex (M1) in participants involved in two reaching conditions: (1) rapid compensatory perturbation-evoked reach to grasp and (2) voluntary reach to grasp in response to an auditory cue. We hypothesized that following cTBS to the left M1 hand area we would find diminished EMG responses in the reaching (right) hand for both compensatory and voluntary movements. To isolate balance reactions to the upper limb participants were seated in an elevated tilt-chair with a stable handle positioned in front of their right shoulder. The chair was held vertical by a magnet and triggered to fall backward randomly. To regain balance, participants were instructed to reach for the handle as quickly as possible with the right hand upon chair release. Intermixed with perturbation trials, participants were also required to reach for the same handle but in response to an auditory tone. Muscle activity was recorded from several muscles of the right arm/hand using electromyography. As expected, movement time and muscle onsets were much faster following perturbation versus auditory-cued reaching. The novel finding from our study was the reduced amplitude of hand muscle activity post-cTBS for both perturbation-cued and auditory-cued reaches. Moreover, this reduction was specific to the

  9. Cross-reactivity of termite myosin; a potential allergen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Myosin and myosin isoforms are common food allergens in crustaceans; such as, shrimp, lobster, and crab. Allergy to Shellfish is a prevalent and potentially long lasting disorder that can severely affect health and quality of life. Myosin and myosin isoforms of dust mites and cockroaches are simil...

  10. Inhibition of the colonic motor response to eating by pinaverium bromide in irritable bowel syndrome patients.

    PubMed

    Fioramonti, J; Frexinos, J; Staumont, G; Bueno, L

    1988-01-01

    The effect of pinaverium bromide on the colonic motor response to eating was investigated in 10 irritable bowel syndrome patients, by means of an intraluminal probe supporting 8 groups of electrodes. At each site examined from transverse to sigmoid colon, the electromyograms exhibited 2 kinds of spike bursts: short spike bursts (SSB) localized at one electrode, and long spike bursts (LSB), isolated, propagated orally or aborally over a few centimeters, or aborally propagated over the whole length of the colon investigated (migrating long spike bursts, MLSB). Recordings were continuously performed over 24 hr. Each patient received at 7.00 p.m. on day 1 and at noon on day 2 an 800-1000 Kcal meal preceded by IV administration of pinaverium bromide (4 mg) or placebo. After placebo administration, the duration of LSB activity and the number of MLSB were significantly increased over 3 postprandial hr by comparison with the 2 hr preceding the meal. After pinaverium injection no significant postprandial change in LSB and MLSB activity was noted. The SSB activity was not modified after the meals preceded by placebo or pinaverium injection. These results suggest that the inhibitory action of pinaverium bromide on postprandial colonic motility may support the clinical efficacy of this agent in the treatment of the irritable bowel syndrome. PMID:3371838

  11. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    SciTech Connect

    Schoenitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  12. Model of myosin recruitment to the cell equator for cytokinesis: feedback mechanisms and dynamical regimes

    NASA Astrophysics Data System (ADS)

    Veksler, Alexander; Vavylonis, Dimitrios

    2011-03-01

    The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During animal cell cytokinesis, cortical myosin filaments (MF) disassemble at the flanking regions and concentrate in the equator. This recruitment depends on myosin motor activity and the Rho proteins that regulate MF assembly and disassembly. Central spindle and astral microtubules help establish a spatial pattern of differential Rho activity. We propose a reaction-diffusion model for the dynamics of MF recruitment to the equatorial region. In the model, the central spindle and mechanical stress promote self-reinforcing MF assembly. Negative feedback is introduced by MF-induced recruitment of inhibitor myosin phosphatase. Our model yields various dynamical regimes and explains both the recruitment of MF to the cleavage furrow and the observed damped MF oscillations in the flanking regions, as well as steady MF assembly. Space and time parameters of MF oscillations are calculated. We predict oscillatory relaxation of cortical MF upon removal of locally-applied external stress.

  13. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

    PubMed

    Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS.

  14. Inhibition of midbrain-evoked tonic and rhythmic motor activity by cutaneous stimulation in decerebrate cats.

    PubMed

    Beyaert, C A; Haouzi, P; Marchal, F

    2003-03-01

    The effect of mechanical and electrical stimulation of cervical cutaneous afferents was analysed on both the centrally induced tonic and rhythmic activities in hindlimb antagonist muscle nerves of 16 decerebrate paralysed cats. Electrical stimulation of dorsal midbrain evoked in the nerve to the tibialis anterior muscle (TAn) either rhythmic discharges (n=14), associated with tonic discharges in ten cats, or only tonic discharges (n=4). Centrally induced activity in the ipsilateral nerve to gastrocnemius medialis (GMn) occurred in fewer cats (n=12) and displayed similar patterns as in TAn. Manual traction of the scruff of the neck reduced the TAn tonic and rhythmic discharges (n=6) by 73% (P<0.05) and 71% (P<0.05), respectively, and reduced only the tonic component of GMn discharges (by 41%, n=3). Electrical stimulation (impulses 0.1-0.5 ms, 50 Hz) of cervical nerves belonging to C5 or C6 dermatomes, the intensity (0.4-4 mA) of which induced minimal inhibition of both TAn and GMn discharges, reduced significantly the tonic component of TAn discharges (by 39%, n=4). At higher intensities of electrical cervical nerve stimulation (2-6 mA) inducing maximal inhibitory effect, both tonic and rhythmic activities in TAn and GMn were both significantly reduced by, respectively, 81% and 94% in TAn (n=7), and by 49% and 43% in GMn (n=7). Electrical cervical nerve stimulation consistently reduced the isolated tonic discharge in TAn by 66% (n=4, P<0.05) and in GMn by 23% (n=3) when present. Thus the tonic component was more sensitive to inhibition than the rhythmic component of hindlimb muscle nerve activity.

  15. Study on the tripolyphosphatase (TPPase) property of bighead carp ( Aristichthys nobilis) myosin subfragment-1

    NASA Astrophysics Data System (ADS)

    Gao, Ruichang; Xue, Changhu; Yuan, Li; Zhang, Yongqin; Xue, Yong; Sun, Yan; Feng, Hui

    2007-10-01

    Myosin subfragment-1 was prepared from the myofibrils of bighead carp ( Aristichthys nobilis). The myosin subfragment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30°C and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2+ was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2+ under low concentrations, but was inhibited when the concentration was over 17 mmolL-1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3 molL-1 under the condition of 17 mmolL-1 Mg2+. The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a K M of 3.2mmolL-1.

  16. Interaction of brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 and kinesin motor protein KIF21A

    PubMed Central

    Shen, Xiaoyan; Meza-Carmen, Victor; Puxeddu, Ermanno; Wang, Guanghui; Moss, Joel; Vaughan, Martha

    2008-01-01

    Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates human ADP-ribosylation factor (ARF) 1 and 3 by accelerating the replacement of ARF-bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Liquid chromatography MS/MS analysis of peptides from proteins that co-precipitated with BIG1 antibodies identified “kinesin family member 21A” (KIF21A), a plus-end-directed motor protein that moves cargo on microtubules away from the microtubule-organizing center. Reciprocal immunoprecipitation (IP) of endogenous proteins and microscopically apparent overlap of immunoreactive BIG1 with overexpressed GFP-KIF21A in the perinuclear region were consistent with an interaction of KIF21A–BIG1. Overexpression of full-length KIF21A and BIG1 and their fragments in HEK293 cells followed by reciprocal IP revealed that the C-terminal tail of KIF21A, with seven WD-40 repeats, may interact with structure in the C-terminal region of BIG1. Interfering with cyclic activation and inactivation of ARF1 by overexpressing constitutively active ARF1(Q71L) or dominant inactive ARF1(T31N) altered the distribution of BIG1 as well as its interaction with KIF21A. A requirement for ARF1 was confirmed by its selective depletion with siRNA. Unlike disruption of microtubules with nocodazole, selective inhibition of transport by depletion of KIF21A with specific siRNA altered BIG1 distribution without changing that of intrinsic Golgi membrane proteins. These newly recognized interactions of BIG1 and KIF21A should enable us to understand better the mechanisms through which, acting together, they may integrate local events in membrane trafficking with longer-range transport processes and to relate those processes to the diverse signaling and scaffold functions of BIG1. PMID:19020088

  17. Mechanical coupling in myosin V: a simulation study.

    PubMed

    Ovchinnikov, Victor; Trout, Bernhardt L; Karplus, Martin

    2010-01-29

    Myosin motor function depends on the interaction between different domains that transmit information from one part of the molecule to another. The interdomain coupling in myosin V is studied with restrained targeted molecular dynamics using an all-atom representation in explicit solvent. To elucidate the origin of the conformational change due to the binding of ATP, targeting forces are applied to small sets of atoms (the forcing sets, FSs) in the direction of their displacement from the rigor conformation, which has a closed actin-binding cleft, to the post-rigor conformation, in which the cleft is open. The "minimal" FS that results in extensive structural changes in the overall myosin conformation is composed of ATP, switch 1, and the nearby HF, HG, and HH helices. Addition of switch 2 to the FS is required to achieve a complete opening of the actin-binding cleft. The restrained targeted molecular dynamics simulations reveal the mechanical coupling pathways between (i) the nucleotide-binding pocket (NBP) and the actin-binding cleft, (ii) the NBP and the converter, and (iii) the actin-binding cleft and the converter. Closing of the NBP due to ATP binding is tightly coupled to the opening of the cleft and leads to the rupture of a key hydrogen bond (F441N/A684O) between switch 2 and the SH1 helix. The actin-binding cleft may mediate the rupture of this bond via a connection between the HW helix, the relay helix, and switch 2. The findings are consistent with experimental studies and a recent normal mode analysis. The present method is expected to be useful more generally in studies of interdomain coupling in proteins.

  18. Unconventional myosin ID is expressed in myelinating oligodendrocytes.

    PubMed

    Yamazaki, Reiji; Ishibashi, Tomoko; Baba, Hiroko; Yamaguchi, Yoshihide

    2014-10-01

    Myelin is a dynamic multilamellar structure that ensheathes axons and is crucial for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes that wrap many layers of plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we found that myosin ID (Myo1d), a class I myosin, is enriched in the rat CNS myelin fraction. Myo1d is an unconventional myosin and has been shown to be involved in membrane trafficking in the recycling pathway in an epithelial cell line. Western blotting revealed that Myo1d expression begins early in myelinogenesis and continues to increase into adulthood. The localization of Myo1d in CNS myelin has not been reported, and the function of Myo1d in vivo remains unknown. To demonstrate the expression of Myo1d in CNS myelin and to begin to explore the function of Myo1d in myelination, we produced a new antibody against Myo1d that has a high titer and specificity for rat Myo1d. By using this antibody, we demonstrated that Myo1d is expressed in rat CNS myelin and is especially abundant in abaxonal and adaxonal regions (the outer and inner cytoplasm-containing loops, respectively), but that expression is low in peripheral nervous system myelin. In culture, Myo1d was expressed in mature rat oligodendrocytes. Furthermore, an increase in expression of Myo1d during maturation of CNS white matter (cerebellum and corpus callosum) was demonstrated by histological analysis. These results suggest that Myo1d may be involved in the formation and/or maintenance of CNS myelin.

  19. The size and the speed of the working stroke of muscle myosin and its dependence on the force

    PubMed Central

    Piazzesi, Gabriella; Lucii, Leonardo; Lombardi, Vincenzo

    2002-01-01

    Myosin II is the motor protein that produces force and shortening in muscle by ATP-driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin II's working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 μs force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head. PMID:12433956

  20. [Role of myosins in depression of sensitivity of Helix neurons to acetylcholine in a cellular analog of habituation].

    PubMed

    Pivovarov, A S; Murzina, G B; Makhnovskiĭ, D A; Vasil'eva, N A; Tret'iakova, M S

    2013-01-01

    We investigated the involvement of cytoskeleton motor proteins, myosins, in the molecular mechanism of sensitivity depression to acetylcholine in Helix command neurons of defensive behavior in a cellular analog of habituation. There were analyzed the effects of several drugs disturbing myosin function: ML-7 and MLCK-IP-18--blockers of myosin light chain kinase, blebbistatin--an inhibitor of non-muscle myosin II, Y-27632--inhibitor of kinases ROCK-I and ROCK-II (activate mainly non-muscle myosin II) on the depression of acetylcholine-induced inward current. It was found that ML-7 and MLCK-IP- 18 weakened current depression; blebbistatin and Y-27632 did not change the depression. The results of experimental inhibitory analysis and mathematical modeling of the effects of inhibitors on the number of membrane-bound cholinergic receptors allow to suggest the involvement ofmyosins (excluding non-muscle myosin II) in the transports of acetylcholine receptors (endo- and exocytosis) that are responsible for sensitivity changes in neuron somatic membrane to acetylcholine in a cellular analog of habituation.

  1. Changes in electrophysiological properties of cat hypoglossal motoneurons during carbachol-induced motor inhibition.

    PubMed

    Fung, S J; Yamuy, J; Xi, M C; Engelhardt, J K; Morales, F R; Chase, M H

    2000-12-01

    The control of hypoglossal motoneurons during sleep is important from a basic science perspective as well as to understand the bases for pharyngeal occlusion which results in the obstructive sleep apnea syndrome. In the present work, we used intracellular recording techniques to determine changes in membrane properties in adult cats in which atonia was produced by the injection of carbachol into the pontine tegmentum (AS-carbachol). During AS-carbachol, 86% of the recorded hypoglossal motoneurons were found to be postsynaptically inhibited on the basis of analyses of their electrical properties; the electrical properties of the remaining 14% were similar to motoneurons recorded during control conditions. Those cells that exhibited changes in their electrical properties during AS-carbachol also displayed large-amplitude inhibitory synaptic potentials. Following sciatic nerve stimulation, hypoglossal motoneurons which responded with a depolarizing potential during control conditions exhibited a hyperpolarizing potential during AS-carbachol. Both spontaneous and evoked inhibitory potentials recorded during AS-carbachol were comparable to those that have been previously observed in trigeminal and spinal cord motoneurons under similar experimental conditions as well as during naturally occurring active sleep. Calculations based on modeling the changes that we found in input resistance and membrane time constant with a three-compartment neuron model suggest that shunts are present in all three compartments of the hypoglossal motoneuron model. Taken together, these data indicate that postsynaptic inhibitory drives are widely distributed on the soma-dendritic tree of hypoglossal motoneurons during AS-carbachol. These postsynaptic inhibitory actions are likely to be involved in the pathophysiology of obstructive sleep apnea. PMID:11102580

  2. Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

    PubMed Central

    Takagi, Yasuharu; Farrow, Rachel E.; Billington, Neil; Nagy, Attila; Batters, Christopher; Yang, Yi; Sellers, James R.; Molloy, Justin E.

    2014-01-01

    Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein’s mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12–14 s−1) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor. PMID:24753602

  3. Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization.

    PubMed

    Yau, Richard G; Peng, Yutian; Valiathan, Rajeshwari R; Birkeland, Shanda R; Wilson, Thomas E; Weisman, Lois S

    2014-03-10

    Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2, binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism that detaches myosin V from select cargoes.

  4. Inhibition of fatty acid oxidation activates transforming growth factor-beta in cerebrospinal fluid and decreases spontaneous motor activity.

    PubMed

    Fujikawa, Teppei; Fujita, Ryo; Iwaki, Yoko; Matsumura, Shigenobu; Fushiki, Tohru; Inoue, Kazuo

    2010-10-01

    We have previously reported that transforming growth factor (TGF)-beta in the cerebrospinal fluid (CSF) is involved in the mechanism underlying the regulation of spontaneous motor activity (SMA) by the central nervous system after exercise. However, it remained unclear what physiological condition triggers the activation of TGF-beta. We hypothesized that the shortage of energy derived from fatty acid (FA) oxidation observed in the early phase of exercise activated TGF-beta in the CSF. To test this hypothesis, we investigated whether mercaptoacetate (MA), an inhibitor of FA oxidation, could induce an activation of TGF-beta in the CSF and a decrease in SMA. Intraperitoneal (i.p.) administration of MA activated TGF-beta in CSF in rats and depressed SMA; 2-deoxyglucose, an inhibitor of carbohydrate oxidation, on the other hand, depressed SMA but failed to activate CSF TGF-beta. Intracisternal administration of anti-TGF-beta antibody abolished the depressive effect of MA on SMA. We also found that the depression of SMA and the activation of TGF-beta in the CSF by i.p. MA administration were eliminated by vagotomy. Our data suggest that TGF-beta in the CSF is activated by the inhibition of FA oxidation via the vagus nerve and that this subsequently induces depression of SMA.

  5. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD. PMID:27440420

  6. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

  7. Single myosin cross-bridge orientation in cardiac papillary muscle detects lever-arm shear strain in transduction.

    PubMed

    Burghardt, Thomas P; Josephson, Matthew P; Ajtai, Katalin

    2011-09-13

    Myosin motors transduce ATP free energy into mechanical work. Transduction models allocate specific functions to motor structural domains beginning with ATP hydrolysis in the active site and ending in a lever-arm rotating power-stroke. Myosin light chains, regulatory (RLC) and essential (ELC), bind IQ-domains on the lever-arm and track its movement. Strong evidence exists that light chains stabilize the lever-arm and that light chain mutation undermines stability. Human ventricular RLC tagged with photoactivatable GFP (HCRLC-PAGFP) replaces native RLC in porcine papillary muscle fibers, restores native contractility, and situates PAGFP for single molecule orientation tracking within the crowded fiber lattice. The spatial emission pattern from single photoactivated PAGFP tagged myosins was observed in z-stacks fitted simultaneously to maximize accuracy in estimated dipole orientation. Emitter dipole polar and azimuthal angle pair scatter plots identified an area where steric and molecular crowding constraints depopulated orientations unfavorable for actin interaction. Transitions between pre- and post-power-stroke states represent the lever-arm trajectory sampled by the data and quantify lever-arm shear strain in transduction at three tension levels. These data identify forces acting on myosin in the in situ fiber system due to crowding, steric hindrance, and actomyosin interaction. They induce lever-arm shear strain observed with single molecule orientation detection. A single myosin work histogram reveals discretized power-stroke substates reminiscent of the Huxley-Simmons model for myosin based contraction [Huxley and Simmons ( 1971 ) Nature 233 , 533]. RLC or ELC mutation, should it impact lever-arm shear strain, will be detected as changes in single myosin shear strain or power-stroke substate distribution.

  8. Dynamic exchange of myosin molecules between thick filaments.

    PubMed

    Saad, A D; Pardee, J D; Fischman, D A

    1986-12-01

    To examine thick filament assembly and myosin exchange, a fluorescence energy transfer assay has been established. Assembly-competent myosin molecules labeled with the sulfhydryl-specific fluorochromes 5-(2-[(iodoacetyl)-amino]ethyl)aminonaphthalene-1-sulfonic acids (IAEDANS) or 5-iodoacetamidofluorescein (IAF) were prepared. Using IAEDANS-labeled myosin as fluorescence donor and IAF-labeled myosin as acceptor, thick filament formation was followed by the decrease in donor fluorescence at 0.1 M KCl/10 mM potassium phosphate, pH 6.9. The critical concentration of myosin--i.e., that concentration that remained unassembled at equilibrium with fully formed filaments--was 40 nM. In FET and 125I-labeled myosin incorporation assays, extensive exchange of myosin between thick filaments was observed. The presence of a critical concentration and the measurements of extensive exchange suggest a dynamic equilibrium between fully polymerized myosin and a small pool of soluble myosin.

  9. Statistical Thermodynamics for Actin-Myosin Binding: The Crucial Importance of Hydration Effects.

    PubMed

    Oshima, Hiraku; Hayashi, Tomohiko; Kinoshita, Masahiro

    2016-06-01

    Actomyosin is an important molecular motor, and the binding of actin and myosin is an essential research target in biophysics. Nevertheless, the physical factors driving or opposing the binding are still unclear. Here, we investigate the role of water in actin-myosin binding using the most reliable statistical-mechanical method currently available for assessing biomolecules immersed in water. This method is characterized as follows: water is treated not as a dielectric continuum but as an ensemble of molecules; the polyatomic structures of proteins are taken into consideration; and the binding free energy is decomposed into physically insightful entropic and energetic components by accounting for the hydration effect to its full extent. We find that the actin-myosin binding brings large gains of electrostatic and Lennard-Jones attractive interactions. However, these gains are accompanied by even larger losses of actin-water and myosin-water electrostatic and LJ attractive interactions. Although roughly half of the energy increase due to the losses is cancelled out by the energy decrease arising from structural reorganization of the water released upon binding, the remaining energy increase is still larger than the energy decrease brought by the gains mentioned above. Hence, the net change in system energy is positive, which opposes binding. Importantly, the binding is driven by a large gain of configurational entropy of water, which surpasses the positive change in system energy and the conformational entropy loss occurring for actin and myosin. The principal physical origin of the large water-entropy gain is as follows: the actin-myosin interface is closely packed with the achievement of high shape complementarity on the atomic level, leading to a large increase in the total volume available to the translational displacement of water molecules in the system and a resultant reduction of water crowding (i.e., entropic correlations among water molecules). PMID

  10. Tumor Stiffness Is Unrelated to Myosin Light Chain Phosphorylation in Cancer Cells

    PubMed Central

    Fry, Madeline; Greene, Madelyne; Chernaya, Olga; Hu, Wen-Yang; Chew, Teng-Leong; Mahmud, Nadim; Kadkol, Shrihari S.; Glover, Sarah; Prins, Gail; Strakova, Zuzana; de Lanerolle, Primal

    2013-01-01

    Many tumors are stiffer than their surrounding tissue. This increase in stiffness has been attributed, in part, to a Rho-dependent elevation of myosin II light chain phosphorylation. To characterize this mechanism further, we studied myosin light chain kinase (MLCK), the main enzyme that phosphorylates myosin II light chains. We anticipated that increases in MLCK expression and activity would contribute to the increased stiffness of cancer cells. However, we find that MLCK mRNA and protein levels are substantially less in cancer cells and tissues than in normal cells. Consistent with this observation, cancer cells contract 3D collagen matrices much more slowly than normal cells. Interestingly, inhibiting MLCK or Rho kinase did not affect the 3D gel contractions while blebbistatin partially and cytochalasin D maximally inhibited contractions. Live cell imaging of cells in collagen gels showed that cytochalasin D inhibited filopodia-like projections that formed between cells while a MLCK inhibitor had no effect on these projections. These data suggest that myosin II phosphorylation is dispensable in regulating the mechanical properties of tumors. PMID:24224004

  11. Interactions of actin, myosin, and an actin-binding protein of chronic myelogenous leukemia leukocytes.

    PubMed Central

    Boxer, L A; Stossel, T P

    1976-01-01

    actin as their major polypeptide component and consistent of tangled thin filaments; (b) Contracted aggregates of cytoplasmic extract gels contained by large quantities of myosin as well as actin; (c) Purified actin-binding protein underwent a temperature-dependent, reversible aggregation and caused low concentrations of purified muscle or CML leukocyte actins to gel in sucrose solutions; (d) The gels formed from purified actin plus purified actin-binding protein slowly contracted in the presence but not in the absence of purified CML leukocyte myosin; (e) Rabbit antiserum against purified CML leukocyte actin-binding protein but not against purified CML leukocyte myosin inhibited the gelation of warmed CML leukocyte extracts. Antiserum against CML leukocyte myosin had no effect on the gelation of CML leukocyte extracts but partially curtailed the contraction of the CML leukocyte extract gels and of gels formed from purified CML leukocyte actin-binding protein plus rabbit skeletal muscle actin. Images PMID:133121

  12. Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1*

    PubMed Central

    Haraguchi, Takeshi; Tominaga, Motoki; Matsumoto, Rie; Sato, Kei; Nakano, Akihiko; Yamamoto, Keiichi; Ito, Kohji

    2014-01-01

    Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg2+-ATPase activity (Vmax = 4 s−1), although their affinities for actin were high (Kactin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s−1, respectively). Physiological concentrations of free Mg2+ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis. PMID:24637024

  13. Running exercise enhances motor functional recovery with inhibition of dendritic regression in the motor cortex after collagenase-induced intracerebral hemorrhage in rats.

    PubMed

    Takamatsu, Yasuyuki; Tamakoshi, Keigo; Waseda, Yuya; Ishida, Kazuto

    2016-03-01

    Rehabilitative approaches benefit motor functional recovery after stroke and relate to neuronal plasticity. We investigated the effects of a treadmill running exercise on the motor functional recovery and neuronal plasticity after collagenase-induced striatal intracerebral hemorrhage (ICH) in rats. Male Wistar rats were injected with type IV collagenase into the left striatum to induce ICH. Sham-operated animals were injected with saline instead of collagenase. The animals were randomly assigned to the sham control (SC), the sham exercise (SE), the ICH control (IC), or the ICH exercise (IE) group. The exercise groups were forced to run on a treadmill at a speed of 9 m/min for 30 min/day between days 4 and 14 after surgery. Behavioral tests were performed using a motor deficit score, a beam-walking test and a cylinder test. At fifteen days after surgery, the animals were sacrificed, and their brains were removed. The motor function of the IE group significantly improved compared with the motor function of the IC group. No significant differences in cortical thickness were found between the groups. The IC group had fewer branches and shorter dendrite lengths compared with the sham groups. However, dendritic branches and lengths were not significantly different between the IE and the other groups. Tropomyosin-related kinase B (TrkB) expression levels increased in the IE compared with IC group, but no significant differences in other protein (brain-derived neurotrophic factor, BDNF; Nogo-A; Rho-A/Rho-associated protein kinase 2, ROCK2) expression levels were found between the groups. These results suggest that improved motor function after a treadmill running exercise after ICH may be related to the prevention of dendritic regression due to TrkB upregulation. PMID:26675889

  14. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    PubMed

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  15. Myosin Heavy Chain Composition of the Human Hyoglossus Muscle*

    PubMed Central

    Sokoloff, Alan J.; Daugherty, Megan; Li, Haiyan

    2013-01-01

    The human tongue muscle hyoglossus (HG) muscle is active in oro-motor behaviors encompassing a wide range of tongue movement speeds. Here we test the hypothesis that the human HG is composed of “uncommon” myosin heavy chain (MHC) isoforms MHCembryonic, MHCneonatal and MHCslow tonic as has been reported for other head and neck muscles active during kinematically diverse behaviors. Following reaction of human HG with antibodies specific for MHCI, MHCIIA, MHCII, MHCembryonic, MHCextraocular, MHCneonatal and MHCslow tonic only antibodies to MHCI, MHCIIA and MHCIIA-X label more than occasional muscle fibers. These antibodies describe five phenotypes with prevalence MHCIIA>MHCI>MHCI-IIX>MHCI-IIA>MHCIIX. In MHC composition, the human HG is thus similar to human appendicular muscles, the human tongue muscle styloglossus and many human head and neck muscles but different from human masseter and extraocular muscles which contain five or more MHC isoforms. PMID:19526266

  16. Sensory Motor Inhibition as a Prerequisite for Theory-of-Mind: A Comparison of Clinical and Normal Preschoolers Differing in Sensory Motor Abilities

    ERIC Educational Resources Information Center

    Chasiotis, Athanasios; Kiessling, Florian; Winter, Vera; Hofer, Jan

    2006-01-01

    After distinguishing between neocortical abilities for executive control and subcortical sensory motor skills for proprioceptive and vestibular integration, we compare a sample of 116 normal preschoolers with a sample of 31 preschoolers receiving occupational therapeutical treatment. This is done in an experimental design controlled for age (mean:…

  17. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    PubMed

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion. PMID:27498562

  18. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    PubMed

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M

    2016-08-22

    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion.

  19. 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.

  20. Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle.

    PubMed

    Tanner, Bertrand C W; McNabb, Mark; Palmer, Bradley M; Toth, Michael J; Miller, Mark S

    2014-06-15

    Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either (i) uniformly, from the Z-line end of thick-filaments, or (ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulations also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere. PMID:24486373

  1. Microfluidic Investigation Reveals Distinct Roles for Actin Cytoskeleton and Myosin II Activity in Capillary Leukocyte Trafficking

    PubMed Central

    Gabriele, Sylvain; Benoliel, Anne-Marie; Bongrand, Pierre; Théodoly, Olivier

    2009-01-01

    Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments. PMID:19450501

  2. Myosin-II sets the optimal response time scale of chemotactic amoeba

    NASA Astrophysics Data System (ADS)

    Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Bodenschatz, Eberhard; Beta, Carsten

    2014-03-01

    The response dynamics of the actin cytoskeleton to external chemical stimuli plays a fundamental role in numerous cellular functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin-II. Here we investigate the role of myosin-II in the response of the actin system to external stimuli. We used a microfluidic device in combination with a photoactivatable chemoattractant to apply stimuli to individual cells with high temporal resolution. We directly compare the actin dynamics in Dictyostelium discodelium wild type (WT) cells to a knockout mutant that is deficient in myosin-II (MNL). Similar to the WT a small population of MNL cells showed self-sustained oscillations even in absence of external stimuli. The actin response of MNL cells to a short pulse of chemoattractant resembles WT during the first 15 sec but is significantly delayed afterward. The amplitude of the dominant peak in the power spectrum from the response time series of MNL cells to periodic stimuli with varying period showed a clear resonance peak at a forcing period of 36 sec, which is significantly delayed as compared to the resonance at 20 sec found for the WT. This shift indicates an important role of myosin-II in setting the response time scale of motile amoeba. Institute of Physics und Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany.

  3. Structural dynamics of the myosin relay helix by time-resolved EPR and FRET

    PubMed Central

    Agafonov, Roman V.; Negrashov, Igor V.; Tkachev, Yaroslav V.; Blakely, Sarah E.; Titus, Margaret A.; Thomas, David D.; Nesmelov, Yuri E.

    2009-01-01

    We have used two complementary time-resolved spectroscopic techniques, dipolar electron–electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeFx) or ADP.Pi (ADP.AlF4) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF4 was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition. PMID:19966224

  4. Chlamydia trachomatis inclusion membrane protein CT228 recruits elements of the myosin phosphatase pathway to regulate release mechanisms

    PubMed Central

    Lutter, Erika I.; Barger, Alexandra C.; Nair, Vinod; Hackstadt, Ted

    2013-01-01

    Summary Chlamydia trachomatis replicates within a membrane bound compartment termed an inclusion. The inclusion membrane is modified by the insertion of multiple proteins known as Incs. In a yeast two-hybrid screen, an interaction was found between the inclusion membrane protein CT228 and MYPT1, a subunit of myosin phosphatase. MYPT1 was recruited peripherally around the inclusion while the phosphorylated inactive form was localized to active Src-family kinase-rich microdomains. Phosphorylated myosin light chain 2 (MLC2), myosin light chain kinase (MLCK), and myosin IIA and IIB also colocalized with inactive MYPT1. The role of these proteins was examined in the context of host-cell exit mechanisms; cell lysis or extrusion of intact inclusions. Inhibition of myosin II or siRNA depletion of myosin IIA and IIB, MLC2, or MLCK reduced chlamydial extrusion thus favoring lytic events as the primary means of release. These studies provide insights into regulation of egress mechanisms by C. trachomatis. PMID:23727243

  5. Alternative S2 Hinge Regions of the Myosin Rod Affect Myofibrillar Structure and Myosin Kinetics

    SciTech Connect

    Miller, Mark S.; Dambacher, Corey M.; Knowles, Aileen F.; Braddock, Joan M.; Farman, Gerrie P.; Irving, Thomas C.; Swank, Douglas M.; Bernstein, Sanford I.; Maughan, David W.

    2009-07-01

    The subfragment 2/light meromyosin 'hinge' region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.

  6. Inhibition of the Motor Protein Eg5/Kinesin-5 in Amyloid β-Mediated Impairment of Hippocampal Long-Term Potentiation and Dendritic Spine Loss.

    PubMed

    Freund, Ronald K; Gibson, Emily S; Potter, Huntington; Dell'Acqua, Mark L

    2016-05-01

    Alzheimer's disease (AD) is characterized by neurofibrillary tangles, amyloid plaques, and neurodegeneration. However, this pathology is preceded by increased soluble amyloid beta (Aβ) 1-42 oligomers that interfere with the glutamatergic synaptic plasticity required for learning and memory, includingN-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). In particular, soluble Aβ(1-42) acutely inhibits LTP and chronically causes synapse loss. Many mechanisms have been proposed for Aβ-induced synaptic dysfunction, but we recently found that Aβ(1-42) inhibits the microtubule motor protein Eg5/kinesin-5. Here we compared the impacts of Aβ(1-42) and monastrol, a small-molecule Eg5 inhibitor, on LTP in hippocampal slices and synapse loss in neuronal cultures. Acute (20-minute) treatment with monastrol, like Aβ, completely inhibited LTP at doses >100 nM. In addition, 1 nM Aβ(1-42) or 50 nM monastrol inhibited LTP #x223c;50%, and when applied together caused complete LTP inhibition. At concentrations that impaired LTP, neither Aβ(1-42) nor monastrol inhibited NMDAR synaptic responses until #x223c;60 minutes, when only #x223c;25% inhibition was seen for monastrol, indicating that NMDAR inhibition was not responsible for LTP inhibition by either agent when applied for only 20 minutes. Finally, 48 hours of treatment with either 0.5-1.0μM Aβ(1-42) or 1-5μM monastrol reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of #x223c;40%, and when applied together at maximal concentrations, no additional spine loss resulted. Thus, monastrol can mimic and in some cases occlude the impact of Aβon LTP and synapse loss, suggesting that Aβinduces acute and chronic synaptic dysfunction in part through inhibiting Eg5.

  7. Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation.

    PubMed

    Dhawan, Jyotsna; Helfman, David M

    2004-08-01

    Cell-substratum interactions trigger key signaling pathways that modulate growth control and tissue-specific gene expression. We have previously shown that abolishing adhesive interactions by suspension culture results in G(0) arrest of myoblasts. We report that blocking intracellular transmission of adhesion-dependent signals in adherent cells mimics the absence of adhesive contacts. We investigated the effects of pharmacological inhibitors of acto-myosin contractility on growth and differentiation of C2C12 myogenic cells. ML7 (5-iodonaphthalene-1-sulfonyl homopiperazine) and BDM (2,3, butanedione monoxime) are specific inhibitors of myosin light chain kinase, and myosin heavy chain ATPase, respectively. ML7 and BDM affected cell shape by reducing focal adhesions and stress fibers. Both inhibitors rapidly blocked DNA synthesis in a dose-dependent, reversible fashion. Furthermore, both ML7 and BDM suppressed expression of MyoD and myogenin, induced p27(kip1) but not p21(cip1), and inhibited differentiation. Thus, as with suspension-arrest, inhibition of acto-myosin contractility in adherent cells led to arrest uncoupled from differentiation. Over-expression of inhibitors of the small GTPase RhoA (dominant negative RhoA and C3 transferase) mimicked the effects of myosin inhibitors. By contrast, wild-type RhoA induced arrest, maintained MyoD and activated myogenin and p21 expression. The Rho effector kinase ROCK did not appear to mediate Rho's effects on MyoD. Thus, ROCK and MLCK play different roles in the myogenic program. Signals regulated by MLCK are critical, since inhibition of MLCK suppressed MyoD expression but inhibition of ROCK did not. Inhibition of contractility suppressed MyoD but did not reduce actin polymer levels. However, actin depolymerization with latrunculin B inhibited MyoD expression. Taken together, our observations indicate that actin polymer status and contractility regulate MyoD expression. We suggest that in myoblasts, the Rho pathway and

  8. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    SciTech Connect

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  9. Cell and molecular biology of the fastest myosins.

    PubMed

    Higashi-Fujime, Sugie; Nakamura, Akio

    2009-01-01

    Chara myosin is a class XI plant myosin in green algae Chara corallina and responsible for fast cytoplasmic streaming. The Chara myosin exhibits the fastest sliding movement of F-actin at 60 mum/s as observed so far, 10-fold of the shortening speed of muscle. It has some distinct properties differing from those of muscle myosin. Although knowledge about Chara myosin is very limited at present, we have tried to elucidate functional bases of its characteristics by comparing with those of other myosins. In particular, we have built the putative atomic model of Chara myosin by using the homology-based modeling system and databases. Based on the putative structure of Chara myosin obtained, we have analyzed the relationship between structure and function of Chara myosin to understand its distinct properties from various aspects by referring to the accumulated knowledge on mechanochemical and structural properties of other classes of myosin, particularly animal and fungal myosin V. We will also discuss the functional significance of Chara myosin in a living cell.

  10. A New Role for Myosin II in Vesicle Fission

    PubMed Central

    Cabeza, Jose M.; Acosta, Jorge; Ramirez-Ponce, Pilar; Ales, Eva

    2014-01-01

    An endocytic vesicle is formed from a flat plasma membrane patch by a sequential process of invagination, bud formation and fission. The scission step requires the formation of a tubular membrane neck (the fission pore) that connects the endocytic vesicle with the plasma membrane. Progress in vesicle fission can be measured by the formation and closure of the fission pore. Live-cell imaging and sensitive biophysical measurements have provided various glimpses into the structure and behaviour of the fission pore. In the present study, the role of non-muscle myosin II (NM-2) in vesicle fission was tested by analyzing the kinetics of the fission pore with perforated-patch clamp capacitance measurements to detect single vesicle endocytosis with millisecond time resolution in peritoneal mast cells. Blebbistatin, a specific inhibitor of NM-2, dramatically increased the duration of the fission pore and also prevented closure during large endocytic events. Using the fluorescent markers FM1-43 and pHrodo Green dextran, we found that NM-2 inhibition greatly arrested vesicle fission in a late phase of the scission event when the pore reached a final diameter of ∼ 5 nm. Our results indicate that loss of the ATPase activity of myosin II drastically reduces the efficiency of membrane scission by making vesicle closure incomplete and suggest that NM-2 might be especially relevant in vesicle fission during compound endocytosis. PMID:24959909

  11. An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

    PubMed Central

    Elangovan, R; Capitanio, M; Melli, L; Pavone, F S; Lombardi, V; Piazzesi, G

    2012-01-01

    A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ. With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4–30°C) and substrate concentration. VF was 8.88 ± 0.26 μm s−1 at 30.6°C and decreased to 1.60 ± 0.09 μm s−1 at 4.5°C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40–50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5°C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads. PMID:22199170

  12. Modes and Mechanisms of T cell Motility: Roles for Confinement and Myosin-IIA

    PubMed Central

    Krummel, Matthew F.; Friedman, Rachel S.; Jacobelli, Jordan

    2014-01-01

    T cells are charged with surveying tissues for evidence of their cognate foreign antigens. Subsequently, they must navigate to effector sites, which they enter through the process of trans-endothelial migration (TEM). During interstitial migration, T cells migrate according to one of two modes that are distinguished by the strength and sequence of adhesions and the requirement for Myosin-IIA. In contrast during TEM, T cells require Myosin-IIA for the final process of pushing their nucleus through the endothelium. A generalized model emerges with dual roles for Myosin-IIA: This motor protein acts like a tensioning or expansion spring, transmitting force across the cell cortex to sites of surface contact and also optimizes the frictional coupling with substrata by modulating the surface area of the contact. The phosphorylation and deactivation of this motor following TCR engagement can allow T cells to rapidly alter the degree to which they adhere to surfaces and to switch to a mode of interaction with surfaces that is more conducive to forming a synapse with an antigen-presenting cell. PMID:24905977

  13. Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Sckolnick, Maria; Bookwalter, Carol S.; Hodges, Alex R.; Trybus, Kathleen M.; Lord, Matthew

    2014-01-01

    A hallmark of class-V myosins is their processivity—the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. PMID:24196839

  14. Myosin-10 and actin filaments are essential for mitotic spindle function

    PubMed Central

    Woolner, Sarah; O'Brien, Lori L.; Wiese, Christiane; Bement, William M.

    2008-01-01

    Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin–based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length. PMID:18606852

  15. Golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-I as a cytoplasmically oriented peripheral membrane protein

    PubMed Central

    1993-01-01

    In the intestinal brush border, the mechanoenzyme myosin-I links the microvillus core actin filaments with the plasma membrane. Previous immunolocalization shows that myosin-I is associated with vesicles in mature enterocytes (Drenckhahn, D., and R. Dermietzel. 1988. J. Cell Biol. 107:1037-1048) suggesting a potential role mediating vesicle motility. We now report that myosin-I is associated with Golgi-derived vesicles isolated from cells that are rapidly assembling brush borders in intestinal crypts. Crypt cells were isolated in hyperosmotic buffer, homogenized, and fractionated using differential- and equilibrium- density centrifugation. Fractions containing 50-100-nm vesicles, a similar size to those observed in situ, were identified by EM and were shown to contain myosin-I as demonstrated by immunoblotting and immunolabel negative staining. Galactosyltransferase, a marker enzyme for trans-Golgi membranes was present in these fractions, as was alkaline phosphatase, which is an apical membrane targeted enzyme. Galactosyltransferase was also present in vesicles immuno-purified with antibodies to myosin-I. Villin, a marker for potential contamination from fragmented microvilli, was absent. Myosin-I was found to reside on the vesicle "outer" or cytoplasmic surface for it was accessible to exogenous proteases and intact vesicles could be immunolabeled with myosin-I antibodies in solution. The bound myosin-I could be extracted from the vesicles using NaCl, KI and Na2CO3, suggesting that it is a vesicle peripheral membrane protein. These vesicles were shown to bundle actin filaments in an ATP-dependent manner. These results are consistent with a role for myosin-I as an apically targeted motor for vesicle translocation in epithelial cells. PMID:8416982

  16. The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head.

    PubMed

    Knight, Peter J; Thirumurugan, Kavitha; Xu, Yuhui; Wang, Fei; Kalverda, Arnout P; Stafford, Walter F; Sellers, James R; Peckham, Michelle

    2005-10-14

    Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke. PMID:16030012

  17. Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

    PubMed Central

    Lecuona, Emilia; Minin, Alexander; Trejo, Humberto E.; Chen, Jiwang; Comellas, Alejandro P.; Sun, Haiying; Grillo, Doris; Nekrasova, Oxana E.; Welch, Lynn C.; Szleifer, Igal; Gelfand, Vladimir I.; Sznajder, Jacob I.

    2009-01-01

    Summary Stimulation of Na+/K+-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na+/K+-ATPase recruitment to the plasma membrane by increasing the average speed of Na+/K+-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na+/K+-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na+/K+-ATPase-containing vesicles, as well as the Na+/K+-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na+/K+-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na+/K+-ATPase to the plasma membrane and thus increased activity. PMID:19808891

  18. 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. PMID:21986200

  19. Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

    PubMed Central

    Oiwa, K; Eccleston, J F; Anson, M; Kikumoto, M; Davis, C T; Reid, G P; Ferenczi, M A; Corrie, J E; Yamada, A; Nakayama, H; Trentham, D R

    2000-01-01

    Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins. PMID:10827983

  20. Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase

    SciTech Connect

    Grishin, M.N.; Kodentsova, V.M.; Abdraimova, U.A.; Nikolaeva, O.P.; Petushkova, E.V.

    1986-03-20

    It was established that the dialdehyde derivative of ATP (oxo-ATP) is a good substrate of the Ca-ATPase of heavy meromyosin: (1.2-1.4) x 10/sup -4/ M; V = V/sub ATP/. At the same time, it is capable of inducing irreversible inhibition of the enzyme. Since oxo-ATP is rapidly digested by myosin, forming oxo-ADP, this inhibition is a consequence of the interaction of the enzyme with oxo-ADP. It was shown that the inhibition of heavy meromyosin (HMM), by oxo-ADP occurs according to the kinetics characteristic of affinity modification; moreover, ATP entirely protects HMM from the loss of activity. Similar data on the irreversible inhibition of ATPase activity under the action of oxo-ADP were obtained in the case of myosin, heavy meromyosin, subfragment-1, and natural actomyosin, as well as in the absence of divalent cations, which is evidence of modification of the active site of myosin ATPase.

  1. Cell rigidity and shape override CD47's "self"-signaling in phagocytosis by hyperactivating myosin-II.

    PubMed

    Sosale, Nisha G; Rouhiparkouhi, Tahereh; Bradshaw, Andrew M; Dimova, Rumiana; Lipowsky, Reinhard; Discher, Dennis E

    2015-01-15

    A macrophage engulfs another cell or foreign particle in an adhesive process that often activates myosin-II, unless the macrophage also engages "marker of self" CD47 that inhibits myosin. For many cell types, adhesion-induced activation of myosin-II is maximized by adhesion to a rigid rather than a flexible substrate. Here we demonstrate that rigidity of a phagocytosed cell also hyperactivates myosin-II, which locally overwhelms self-signaling at a phagocytic synapse. Cell stiffness is one among many factors including shape that changes in erythropoiesis, in senescence and in diseases ranging from inherited anemias and malaria to cancer. Controlled stiffening of normal human red blood cells (RBCs) in different shapes does not compromise CD47's interaction with the macrophage self-recognition receptor signal regulatory protein alpha (SIRPA). Uptake of antibody-opsonized RBCs is always fastest with rigid RBC discocytes, which also show that maximal active myosin-II at the synapse can dominate self-signaling by CD47. Rigid but rounded RBC stomatocytes signal self better than rigid RBC discocytes, highlighting the effects of shape on CD47 inhibition. Physical properties of phagocytic targets thus regulate self signaling, as is relevant to erythropoiesis, to clearance of rigid RBCs after blood storage, clearance of rigid pathological cells such as thalassemic or sickle cells, and even to interactions of soft/stiff cancer cells with macrophages.

  2. Myosin translocation in retinal pericytes during free-radical induced apoptosis.

    PubMed

    Shojaee, N; Patton, W F; Hechtman, H B; Shepro, D

    1999-10-01

    Vascular pathologies induced by ischemia/reperfusion involve the production of reactive oxygen species (ROS) that in part cause tissue injury. The production of ROS that occurs upon reperfusion activates specific second messenger pathways. In diabetic retinopathy there is a characteristic loss of the microvascular pericyte. Pericytes are more sensitive than endothelial cells to low concentrations of ROS, such as hydrogen peroxide (H(2)O(2)) when tested in vitro. Whether the pericyte loss is due to toxic cell death triggered by the noxious H(2)O(2) or apoptosis, due to activation of specific second messenger pathways, is unknown. During apoptosis, a cell's nucleus and cytoplasm condense, the cell becomes fragmented, and ultimately forms apoptotic bodies. It is generally assumed that apoptosis depends on nuclear signaling, but cytoplasmic morphological processes are not well described. We find that exposing cultured retinal pericytes to 100 microM H(2)O(2) for 30 min leads to myosin heavy chain translocation from the cytosol to the cytoskeleton and a significant decrease in cell surface area. Pericyte death follows within 60-120 min. Exposing cells to 150 mJ/cm(2) ultraviolet radiation, an alternate free radical generating system, also causes pericyte myosin translocation and apoptosis. Proteolytic cleavage of actin is not observed in pericyte apoptosis. 3-aminobenzamide, a pharmacological inhibitor of the cleavage and activation of the DNA-repairing enzyme poly (ADP-ribose) polymerase (PARP) inhibits pericyte apoptosis, and prevents myosin translocation. Deferoxamine, an iron chelator known to interfere with free radical generation, also inhibits pericyte myosin translocation, contractility, and cell death. Myosin translocation to the cytoskeleton may be an early step in assembly of a competent contractile apparatus, which is involved in apoptotic cell condensation. These results suggest that pericyte loss associated with increased free radical production in

  3. Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments.

    PubMed

    Suzuki, Makoto; Kabir, Syed Rashel; Siddique, Md Shahjahan Parvez; Nazia, Umme Salma; Miyazaki, Takashi; Kodama, Takao

    2004-09-10

    Microwave dielectric spectroscopy can measure the rotational mobility of water molecules that hydrate proteins and the hydration-shell volume. Using this technique, we have recently shown that apart from typical hydrating water molecules with lowered mobility there are other water molecules around the actin filaments (F-actin) which have a much higher mobility than that of bulk water [Biophys. J. 85 (2003) 3154]. We report here that the volume of this water component (hyper-mobile water) markedly increases without significant change of the volume of the ordinary hydration shell when the myosin motor-domain (S1, myosin subfragment-1) binds to F-actin. No hyper-mobile component was found in the hydration shell of S1 itself. The present results strongly suggest that the solvent space around S1 bound to F-actin is diffusionally asymmetric, which supports our model of force generation by actomyosin proposed previously [op. cit.]. PMID:15313212

  4. Preparation and Characterization of Myosin Proteins.

    ERIC Educational Resources Information Center

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  5. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  6. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.

    PubMed

    Wang, Xueqian; Chong, Mei; Wang, Xin; Wang, Hongkui; Zhang, Jie; Xu, Hui; Zhang, Jingjing; Liu, Dong

    2015-03-01

    Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro.

  7. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.

    PubMed

    Wang, Xueqian; Chong, Mei; Wang, Xin; Wang, Hongkui; Zhang, Jie; Xu, Hui; Zhang, Jingjing; Liu, Dong

    2015-03-01

    Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro. PMID:25403653

  8. A striking common O-linked N-acetylglucosaminyl moiety between cruzipain and myosin.

    PubMed

    Acosta, D M; Soprano, L L; Ferrero, M; Landoni, M; Esteva, M I; Couto, A S; Duschak, V G

    2011-07-01

    Single units of O-linked N-acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C-terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self-antigens like myosin are associated with Chagas heart disease. The participation of O-GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross-reactivity between Cz and myosin is mainly focused in the C-T domain. ELISA inhibition assays using rabbit sera specific for Cz and C-T in conjunction with immune-gold electron microscopy analysis of heart tissues from mice immunized with C-T confronted with polyclonal rabbit sera specific for Cz and C-T prior and after myosin adsorption provided evidence which indicates that O-GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O-GlcNAc-containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease. PMID:21426361

  9. Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

    PubMed Central

    Ueda, Shuji; Blee, Alexandra M.; Macway, Katherine G.; Renner, Derrick J.; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  10. Myosin 1e is a component of the invadosome core that contributes to regulation of invadosome dynamics

    PubMed Central

    Ouderkirk, Jessica L.; Krendel, Mira

    2014-01-01

    Myosin 1e (myo1e) is an actin-based motor protein that has been implicated in cell adhesion and migration. We examined the role of myo1e in invadosomes, actin-rich adhesion structures that are important for degradation and invasion of the extracellular matrix. RSV-transformed BHK-21 cells, which readily form invadosomes and invadosome rosettes, were used as the experimental model. Myo1e localization to the actin-rich core of invadosomes required the proline-rich Tail Homology 2 (TH2) domain. During invadosome rosette expansion, we observed myo1e recruitment to newly forming invadosomes via Tail Homology 1 (TH1)-dependent interactions with the plasma membrane, where it preceded actin and paxillin. Dominant-negative inhibition of myo1e resulted in mislocalized invadosome formation, usually at the center of the rosette. We propose that TH2 domain of myo1e provides the key signal for localization to invadosomes, while TH1 domain interactions facilitate myo1e targeting to the plasma membrane-proximal locations within the rosettes. Myo1e may then act as a scaffold, linking the plasma membrane with the actin cytoskeleton and helping direct new invadosome formation to the periphery of the rosette. PMID:24462457

  11. Molecular motors and their functions in plants

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.

    2001-01-01

    Molecular motors that hydrolyze ATP and use the derived energy to generate force are involved in a variety of diverse cellular functions. Genetic, biochemical, and cellular localization data have implicated motors in a variety of functions such as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. In non-plant systems three families of molecular motors (kinesins, dyneins, and myosins) have been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials intracellularly. During the last decade tremendous progress has been made in understanding the structure and function of various motors in animals. These studies are yielding interesting insights into the functions of molecular motors and the origin of different families of motors. Furthermore, the paradigm that motors bind cargo and move along cytoskeletal tracks does not explain the functions of some of the motors. Relatively little is known about the molecular motors and their roles in plants. In recent years, by using biochemical, cell biological, molecular, and genetic approaches a few molecular motors have been isolated and characterized from plants. These studies indicate that some of the motors in plants have novel features and regulatory mechanisms. The role of molecular motors in plant cell division, cell expansion, cytoplasmic streaming, cell-to-cell communication, membrane trafficking, and morphogenesis is beginning to be understood. Analyses of the Arabidopsis genome sequence database (51% of genome) with conserved motor domains of kinesin and myosin families indicates the presence of a large number (about 40) of molecular motors and the functions of many of these motors remain to be discovered. It is likely that many more motors with novel regulatory

  12. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    SciTech Connect

    Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  13. Myosin Heavy Chain Composition of the Human Genioglossus Muscle

    PubMed Central

    Daugherty, Megan; Luo, Qingwei; Sokoloff, Alan J.

    2013-01-01

    Background The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation but whether this is accompanied by complex expression of muscle contractile proteins is not known. Purpose We tested for conventional myosin heavy chain MHCI, MHCIIA, MHCIIX, developmental MHCembryonic and MHCneonatal and unconventional MHCαcardiac, MHCextraocular and MHCslow tonic in antero-superior (GG-A) and posterior (GG-P) adult human GG. Method SDS-PAGE, Western blot and immunohistochemistry were used to describe MHC composition of GG-A and GG-P and the prevalence of muscle fiber MHC phenotypes in GG-A. Results: By SDS-PAGE, only conventional MHC are present with ranking from most to least prevalent MHCIIA>MHCI>MHCIIX in GG-A and MHCI>MHCIIA>MHCIIX in GG-P. By immunohistochemistry many muscle fibers contain MHCI, MHCIIA and MHCIIX but few contain developmental or unconventional MHC. GG-A is composed of five phenotypes (MHCIIA>MHCI-IIX>MHCI>MHCI-IIA>MHCIIX). Phenotypes MHCI, MHCIIA and MHCI-IIX account for 96% of muscle fibers. Conclusions Despite activation of GG during kinematically diverse behaviors and complex patterns of GG motor unit activity, the human GG is composed of conventional MHC isoforms and three primary MHC phenotypes. PMID:22337492

  14. Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots

    PubMed Central

    Kakizuka, Taishi; Ikezaki, Keigo; Kaneshiro, Junichi; Fujita, Hideaki; Watanabe, Tomonobu M.; Ichimura, Taro

    2016-01-01

    Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery. PMID:27446684

  15. Molecular Motors: A Theorist's Perspective

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.; Fisher, Michael E.

    2007-05-01

    Individual molecular motors, or motor proteins, are enzymatic molecules that convert chemical energy, typically obtained from the hydrolysis of ATP (adenosine triphosphate), into mechanical work and motion. Processive motor proteins, such as kinesin, dynein, and certain myosins, step unidirectionally along linear tracks, specifically microtubules and actin filaments, and play a crucial role in cellular transport processes, organization, and function. In this review some theoretical aspects of motor-protein dynamics are presented in the light of current experimental methods that enable the measurement of the biochemical and biomechanical properties on a single-molecule basis. After a brief discussion of continuum ratchet concepts, we focus on discrete kinetic and stochastic models that yield predictions for the mean velocity, V(F, [ATP], …), and other observables as a function of an imposed load force F, the ATP concentration, and other variables. The combination of appropriate theory with single-molecule observations should help uncover the mechanisms underlying motor-protein function.