Science.gov

Sample records for actin-activated myosin atpase

  1. Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

    PubMed

    Muretta, Joseph M; Petersen, Karl J; Thomas, David D

    2013-04-30

    We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production. PMID:23589853

  2. 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

  3. 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

  4. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    SciTech Connect

    Vu, N.D.; Wagner, P.D.

    1987-07-28

    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of /sup 32/P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca/sup 2 +/- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation.

  5. 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

  6. 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.

  7. Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.

    PubMed

    Hiratsuka, T

    1992-07-25

    The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant S-1 derivative (MIANS-S-1) was enhanced by ATP in a manner similar to that of unlabeled S-1. The quenching of tryptophan fluorescence of MIANS-S-1 was shown to result from a transfer of the excitation energy from tryptophanyl residue(s) to the MIANS fluorophore attached to SH2, which absorbed and fluoresced maximally at 325 and 418 nm, respectively. The energy transfer measurements were performed in the presence of acrylamide and compared to those performed in the absence of the quencher. The energy transfer efficiencies were found to be unaltered by acrylamide, indicating that the observed fluorescence energy transfer is originated exclusively from the tryptophanyl residue(s) that are not affected by acrylamide, i.e. the ATP-sensitive tryptophanyl residue(s) of S-1 (Torgerson, P. M. (1984) Biochemistry 23, 3002-3007). The distance between the tryptophanyl residue(s) and Cys-697 was calculated to be 27 A assuming a single donor-acceptor pair. Trp-510 is proposed to be one of the ATP-sensitive tryptophanyl residues. PMID:1386083

  8. Ca2+- activated myosin- ATPases, creatine and adenylate kinases regulate mitochondrial function according to myofibre type in rabbit

    PubMed Central

    Gueguen, N; Lefaucheur, L; Ecolan, P; Fillaut, M; Herpin, P

    2005-01-01

    Mitochondrial respiration rates and their regulation by ADP, AMP and creatine, were studied at different free Ca2+ concentrations (0.1 versus 0.4 μm) on permeabilized fibre bundles of rabbit skeletal muscles differing in their myosin heavy chain profiles. Four fibre bundle types were obtained: pure types I and IIx, and mixed types IIax (approximately 50% IIa and 50% IIx fibres) and IIb+ (60% IIb fibres, plus IIx and IIa). At rest, pure type I fibres displayed a much higher apparent Km for ADP (212 μm) than IIx fibres (8 μm). Within the IIax and IIb+ mixed fibre bundle types, two KADPm values were observed (70 μm and 5 μm). Comparison between pure IIx and mixed types indicates that the intermediate Km of 70 μm most probably corresponds to the mitochondrial affinity for ADP in IIa fibres, the lowest Km for ADP (5 μm) corresponding to IIx and IIb types. Activation of mitochondrial creatine and adenylate kinase reactions stimulated mitochondrial respiration only in type I and IIax fibre bundles, indicating an efficient coupling between both kinases and ADP rephosphorylation in type I and, likely, IIa fibres, since no effect was observed in pure IIx fibres. Following Ca2+-induced activation of myosin-ATPase, an increase in mitochondrial sensitivity to ADP of 45% and 250% was observed in type IIax and I bundles, respectively, an effect mostly prevented by addition of vanadate, an inhibitor of myosin-ATPase. Ca2+-induced activation of myosin-ATPase also prevented the stimulation of respiration rates by creatine and AMP in I and IIax bundles. In addition to differential regulation of mitochondrial respiration and energy transfer systems at rest in I and IIa versus IIx and IIb muscle fibres, our results indicate a regulation of phosphotransfer systems by Ca2+ via the stimulation of myosin-ATPases in type I and IIa fibres of rabbit muscles. PMID:15731190

  9. 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

  10. 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

    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

  11. Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

    PubMed Central

    Sutoh, K; Yamamoto, K; Wakabayashi, T

    1986-01-01

    An ADP analog carrying a biotin moiety and a photoreactive group was synthesized. In the presence of vanadate ion (Vi), the analog was tightly trapped into the ATPase site of heavy meromyosin (HMM) or myosin subfragment 1 (S1) in an ADP analog/ATPase site molar ratio of 1:1. UV illumination on the HMM (or S1)-Vi-ADP analog complex resulted in covalent incorporation of the analog into the ATPase site. About 15% of the trapped analog was crosslinked to HMM or S1. Mapping of the crosslinking site of the analog showed that the N-terminal Mr 25,000 segment of the heavy chain participated in binding the ADP analog. The biotin moiety of the analog covalently incorporated into the ATPase site was visualized in electron microscopy by attaching an avidin oligomer. Rotary-shadowed images of the HMM-avidin complex revealed that the crosslinked ADP analog was located about 140 A from the head-rod junction on the head. The result indicates that the ATPase site of myosin is about 140 A apart from the head-rod junction along the head. Images PMID:2934740

  12. Inhibiting Myosin-ATPase Reveals Dynamic Range of Mitochondrial Respiratory Control in Skeletal Muscle

    PubMed Central

    Perry, Christopher G.R.; Kane, Daniel A.; Lin, Chien-Te; Kozy, Rachel; Cathey, Brook L.; Lark, Daniel S.; Kane, Constance L.; Brophy, Patricia M.; Gavin, Timothy P; Anderson, Ethan J.; Neufer, P. Darrell

    2013-01-01

    Assessment of mitochondrial ADP-stimulated respiratory kinetics in permeabilized skeletal myofibres (PmFB) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (?20-300 ?M) and tend to overestimate respiration at rest. Noting PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. Blebbistatin (BLEB), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >?250 and ?80 ?M in red and white rat PmFB, respectively. In the absence of BLEB, PmFB contracted and the Km for ADP decreased by ?2 to 10-fold in a temperature-dependent manner. PmFB were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30C but not 37C. In PmFB from humans, contraction elicited high sensitivity to ADP (m <100 ?M) whereas blocking contraction (+BLEB) and including PCr:Cr = 2 to mimic the resting energetic state yielded a Km for ADP = ?1560 ?M, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state. PMID:21554250

  13. Mechanism for coupling free energy in ATPase to the myosin active site.

    PubMed

    Park, S; Ajtai, K; Burghardt, T P

    1997-03-18

    Acrylamide quenching of tryptophan 510 (Trp510) fluorescence in rabbit skeletal myosin subfragment 1 (S1) indicates the conformation of the probe binding cleft, containing the highly reactive thiol (SH1) and Trp510, in the presence of nucleotides or nucleotide analogs trapped in the active site of S1 [Park et al. (1996) Biochim. Biophys. Acta 1296, 1-4]. The Trp510 quenching efficiency shows that the probe binding cleft closes slightly in the presence of beryllium fluoride trapped MgADP (MgADPBeFx-S1) and most tightly in the presence of vanadate trapped MgADP (MgADPVi-S1) with aluminum fluoride and scandium fluoride trapped MgADP (MgADPA1F4-S1 and MgADPScFx-S1) falling in between in the order MgADPBeFx > MgADPA1F4 > MgADPScFx > MgADPVi. These nucleotide analogs are identified with sequential structural changes in MgATP during hydrolysis in the same order with beryllium fluoride occurring earliest in the ATPase cycle. Correlation of the separation distance of the gamma-phosphate analog metal from the oxygen connecting it to the beta-phosphate in ADP, to the extent of cleft closure, suggests that this distance in the nucleotide transition state determines the conformation of the probe binding cleft. Trp510 quenching efficiency was also measured as a function of the base moiety of the vanadate trapped Mg-nucleotide diphosphate (MgNDPVi-S1). The extent of cleft closure is largest in the presence of the natural substrate NDP and follows the order MgADPVi > MgCDPVi > MgUDPVi > MgIDPVi > MgGDPVi with very little difference between MgADPVi and MgCDPVi. These data follow the order of the effectiveness of the corresponding nucleotide triphosphates to support force production in muscle fibers [Pate et al. (1993) J. Biol. Chem. 268, 10046-10053]. In both the fiber and S1, it appears that the 6-position amino group of the bases of ADP and CDP is required to properly anchor the nucleotide in the active site, possibly at tyrosine 135 as suggested by X-ray crystallographic studies [Fisher et al. (1995) Biochemistry 34, 8960-8972]. Finally, the Trp510 quenching efficiency was measured as a function of the size of the divalent cation trapped in the active site of S1 with ADPVi. These data failed to show a correlation suggesting that the divalent cation is not involved with the propagation of influence from the active site to the probe binding cleft. The forgoing experiments suggest that the changing conformation of ATP during hydrolysis, parameterized by the increasing distance between the beta- and the gamma-phosphate, stresses the active site of S1 through protein-nucleotide contacts at the gamma-phosphate and nucleotide base. The stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure. PMID:9116016

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

    PubMed Central

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

    2012-01-01

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

  15. 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.

  16. Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity.

    PubMed

    Chou, Y H; Rebhun, L I

    1986-04-25

    The crude actomyosin precipitate from sea urchin (Arbacia punctulata) egg extracts contains Ca2+-sensitive myosin light chain kinase activity. Activity can be further increased by exogenous calmodulin (CaM). Egg myosin light chain kinase activity is purified from total egg extract by fractionating on three different chromatographic columns: DEAE ion exchange, gel filtration on Sephacryl-300, and Affi-Gel-CaM affinity. The purified egg kinase depends totally on Ca2+ and CaM for activity. Unphosphorylated egg myosin has very little actin-activated ATPase. After phosphorylation of the phosphorylable light chain by either egg kinase or gizzard myosin light chain kinase, the actin-activated ATPase of egg myosin is enhanced several fold. However, the egg kinase bears some unique characteristics which are very different from conventional myosin light chain kinases of differentiated tissues. The purified egg kinase has a native molecular mass of 405 kDa, while on sodium dodecyl sulfate-polyacrylamide electrophoresis it shows a single subunit of 56 kDa. The affinity of egg kinase for CaM (Ka = 0.4 microM) is relatively weaker than that of the gizzard myosin light chain kinase. The egg kinase autophosphorylates in the presence of Ca2+ and CaM and has a rather broad substrate specificity. The possible relationship between this egg Ca2+-CaM-dependent kinase and the Ca2+-CaM-dependent kinases from brain and liver is discussed. PMID:2937787

  17. SH-1 modification of rabbit myosin interferes with calcium regulation.

    PubMed

    Titus, M A; Ashiba, G; Szent-Györgyi, A G

    1989-02-01

    The reactive thiol of the myosin head, SH-1, can be selectively labelled in glycerinated rabbit muscle fibres. This residue has been used as an attachment site for either fluorescent or spectroscopic probes which report on head movements and orientations in various functional states of muscle. We have specifically modified SH-1 in vitro, using purified rabbit myosin and conditions similar to those employed in the labelling of muscle fibres (low ionic strength [40 mM NaCl] at 4 degrees C), with stoichiometric amounts of either [14C]-iodoacetamide, 5-(2[iodoacetyl)amino)ethyl) aminonaphthalene-1- sulphonic acid (IAEDANS), or 4-(2-iodoacetamido-2,2,6,6-tetramethyl piperidinooxyl (IASL). The specificity of modification was determined by measuring the well-defined alterations in the high salt ATPase activities of myosin and by localizing both IAAm and IAEDANS to the 20-kDa C-terminal subfragment 1 (S1) which contains SH-1. The low ionic strength actin-activated Mg2+-ATPase of SH-1-modified rabbit myosin was measured in the presence of the thin filament regulatory, complex, troponin-tropomyosin. A significant increase in this activity in the absence of calcium, concomitant with a decrease in activity in the presence of calcium, was observed as the extent of SH-1 modification was incrementally increased from zero to one mole of label bound per mole of SH-1. The elevated myosin Mg2+-ATPase, which results from SH-1 modification, does not account for the increased actin-activated Mg2+-ATPase in resting conditions (i.e. in the absence of calcium).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2523409

  18. 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

  19. Erythrocyte Protein 4.1 Binds and Regulates Myosin

    NASA Astrophysics Data System (ADS)

    Pasternack, Gary R.; Racusen, Richard H.

    1989-12-01

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

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

    PubMed Central

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

    2014-01-01

    Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle. PMID:24578340

  1. Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.

    PubMed

    Hiratsuka, T

    1992-07-25

    To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173). PMID:1386082

  2. 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

  3. 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. PMID:25754640

  4. Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase.

    PubMed Central

    Walker, M; Trinick, J; White, H

    1995-01-01

    The structure of the AM-ATP transient kinetic state of the acto-myosin ATPase cycle has been examined by electron microscopy using frozen-hydrated specimens prepared in low ionic strength. By spraying grids layered with the acto-S1 complex with ATP immediately before freezing, it was possible to examine the structure of the ternary complex with a time resolution of 10 ms. Disordered binding of the S1 was observed, suggesting more than one attachment geometry. This could be due to the presence of more than one biochemical intermediate, or to a single intermediate binding in more than one conformation. Images FIGURE 2 PMID:7787114

  5. A Failure to Communicate: MYOSIN RESIDUES INVOLVED IN HYPERTROPHIC CARDIOMYOPATHY AFFECT INTER-DOMAIN INTERACTION.

    PubMed

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

    2015-12-01

    Our molecular modeling studies suggest a charge-dependent interaction between residues Glu-497 in the relay domain and Arg-712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (~80%) as well as in basal (~67%) and actin-activated (~84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ~73% of wild-type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. Although sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Furthermore, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease. PMID:26446785

  6. Constitutive activation of endocytosis by mutation of myoA, the myosin I gene of Aspergillus nidulans.

    PubMed

    Yamashita, R A; May, G S

    1998-06-01

    Class I myosins function in cell motility, intracellular vesicle trafficking and endocytosis. Recently, it was shown that class I myosins are phosphorylated by a member of the p21-activated kinase (PAK) family. PAK phosphorylates a conserved serine or threonine residue in the myosin heavy chain. Phosphorylation at this site is required for maximal activation of the actin-activated Mg2+-ATPase activity in vitro. This serine or threonine residue is conserved in all known class I myosins of microbial origin and in the human and mouse class VI myosins. We have investigated the in vivo significance of this phosphorylation by mutating serine 371 of the class I myosin heavy chain gene myoA of Aspergillus nidulans. Mutation to glutamic acid, which mimics phosphorylation and therefore activation of the myosin, results in an accumulation of membranes in growing hyphae. This accumulation of membranes results from an activation of endocytosis. In contrast, mutation of serine 371 to alanine had no discernible effect on endocytosis. These studies are the first to demonstrate the in vivo significance of a regulatory phosphorylation on a class I myosin. Furthermore, our results suggest that MYOA has two functions, one dependent and one independent of phosphorylation. PMID:9603982

  7. Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants

    PubMed Central

    LINNANE, LINDA; SERRANO, A. L.; RIVERO, J. L. L.

    1999-01-01

    The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of ‘pure’ I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling site. Within the fast fibres, types IIA and IIAX MHC-classified fibres were proportionately more abundant in the deepest sampling site than in the superficial region of the muscle. The immunohistochemical and histochemical characterisation of a large number of single fibres (n=1375) was compared and correlated on a fibre-to-fibre basis. The results showed that 40% of the fibres analysed were pure type I (expressing only MHC-I); they showed correct matching between their antigenic and mATPase determinants. In contrast, within the fast fibres, a considerable proportion of fibres were found showing a mismatch between their immunohistochemical and mATPase profiles. The most common mismatched fibre phenotypes comprised fibres displaying coexpression of both fast MHCs when analysed by immunocytochemistry, but showing an mATPase profile similar to typical IIX fibres (moderate mATPase reaction after preincubation at pH 4.4). Considered altogether, the total mismatched fibres represented only 4.2% of the whole fast fibre population in the superficial region of the muscle, but their proportion increased to 15.6% and 38.4% in the middle and deep regions, respectively, of gluteus medius. It is concluded that a considerable number of hybrid fast MHC IIAX fibres are present in the gluteus medius of untrained horses, suggesting that equine type II fibres have probably been misclassified in numerous previous publications based on the use of histochemistry alone. This has important implications in attempts to study the physiological properties of fast fibre types adequately in horses. PMID:10386774

  8. The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro*

    PubMed Central

    Haldeman, Brian D.; Brizendine, Richard K.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

    2014-01-01

    Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ∼0.63 μm long and contain ∼176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment-limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment-limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

  9. The kinetics underlying the velocity of smooth muscle myosin filament sliding on actin filaments in vitro.

    PubMed

    Haldeman, Brian D; Brizendine, Richard K; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

    2014-07-25

    Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ~0.63 μm long and contain ~176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment- limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment- limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

  10. Correlation of myosin isoenzyme alterations with myocardial function in physiologic and pathologic hypertrophy.

    PubMed

    Malhotra, A; Schaible, T F; Capasso, J; Scheuer, J

    1984-12-01

    To explore the interactions of physiologic and pathologic hypertrophy, four groups of hearts were studied in an isolated working rat heart apparatus. Cardiac contractile proteins were also evaluated. The groups were hearts of female control sedentary rats; rats subjected to a 10-week swimming programme; rats with renal hypertension; and hypertensive rats subjected to a 10-week swimming programme. The swimming programme in normotensive female rats caused a 30% cardiac hypertrophy, in hypertensive animals 46% hypertrophy, and in combined hypertension and swimming 70% hypertrophy. Ca2+-myosin ATPase activity and actin-activated myosin ATPase were elevated in hearts of swimmers, depressed in hearts of hypertensive sedentary animals and similar to control values in hearts of hypertensive swimmers. Myosin V1 isoenzyme content was increased in hearts of swimmers, depressed in hearts of hypertensives, but normal in hearts of hypertensive swimmers. Reciprocal relationships were seen with the V3 isoenzyme. Stroke work, mean velocity of circumferential fibre shortening, and per cent fractional shortening at the midwall showed increased values for hearts of swimmers, depressed values for hearts of hypertensives, and normal values or values above the control for hearts of hypertensive swimmers. Myocardial flow measured with microspheres was increased in the left ventricle of swimmers, depressed in hearts of hypertensives and still depressed in hypertensive swimmers, but significantly higher than in the hypertensives alone. The correlation of actin-activated ATPase activity and of fractional shortening was linear among the four groups. These studies demonstrate that physiologic and pathologic hypertrophy in the rat have distinctly opposite effects on contractile proteins and contractile performance. When one type of hypertrophy is superimposed on the other the effects are additive. PMID:6241904

  11. UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

    PubMed Central

    Lord, Matthew; Pollard, Thomas D.

    2004-01-01

    We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis. PMID:15504913

  12. Effects of chronic growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin pattern, and myosin adenosine triphosphatase activity of rat left ventricle.

    PubMed Central

    Timsit, J; Riou, B; Bertherat, J; Wisnewsky, C; Kato, N S; Weisberg, A S; Lubetzki, J; Lecarpentier, Y; Winegrad, S; Mercadier, J J

    1990-01-01

    We studied papillary muscle mechanics and energetics, myosin phenotype, and ATPase activities in left ventricles from rats bearing a growth hormone (GH)--secreting tumor. 18 wk after tumor induction, animals exhibited a dramatic increase in body weight (+101% vs. controls) but no change in the ventricular weight/body weight ratio. The maximum isometric force of papillary muscles normalized per cross-sectional area rose markedly (+42%, P less than 0.05 vs. controls), whereas the maximum unloaded shortening velocity did not change. This was observed despite a marked isomyosin shift towards V3 (32 +/- 5% vs. 8 +/- 2% in controls, P less than 0.001). Increased curvature of the force-velocity relationship (+64%, P less than 0.05 vs. controls) indicated that the muscles contracted more economically, suggesting the involvement of V3 myosin. Total calcium- and actin-activated myosin ATPase activities assayed on quickly frozen left ventricular sections were similar in tumor-bearing rats and in controls. After alkaline preincubation, these activities only decreased in tumor-bearing rats, demonstrating that V3 enzymatic sites were involved in total ATPase activity. These data demonstrate that chronic GH hypersecretion in the rat leads to a unique pattern of myocardial adaptation which allows the muscle to improve its contractile performance and economy simultaneously, thanks to myosin phenoconversion and an increase in the number of active enzymatic sites. Images PMID:2143510

  13. Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges

    PubMed Central

    Brizendine, Richard K.; Alcala, Diego B.; Carter, Michael S.; Haldeman, Brian D.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

    2015-01-01

    It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley’s 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255–318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197–218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d = 8 nm gave an ADP release rate 8.5-fold faster and ton (myosin’s attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V = N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V = L/ton rather than d/ton, where L is related to the length of myosin’s subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d = 11.1 nm. Therefore, in contrast with Huxley’s model, we conclude that V0 is limited by the actin–myosin attachment rate. PMID:26294254

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  16. Structure and Regulation of the Movement of Human Myosin VIIA.

    PubMed

    Sakai, Tsuyoshi; Jung, Hyun Suk; Sato, Osamu; Yamada, Masafumi D; You, Dong-Ju; Ikebe, Reiko; Ikebe, Mitsuo

    2015-07-10

    Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca(2+). Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca(2+), and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition. PMID:26001786

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

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki; Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K{sub app} reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.

  18. Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography.

    PubMed

    Liu, Jun; Taylor, Dianne W; Krementsova, Elena B; Trybus, Kathleen M; Taylor, Kenneth A

    2006-07-13

    Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion. PMID:16625208

  19. Cooperation between the two heads of smooth muscle myosin is essential for full activation of the motor function by phosphorylation.

    PubMed

    Ma, Rong-Na; Mabuchi, Katsuhide; Li, Jing; Lu, Zekuan; Wang, Chih-Lueh Albert; Li, Xiang-dong

    2013-09-10

    The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM. PMID:23947723

  20. 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)

  1. 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. PMID:22648562

  2. Germinal Center Specific Protein HGAL Directly Interacts with Both Myosin and Actin and Increases the Binding of Myosin to Actin

    PubMed Central

    Lu, Xiaoqing; Kazmierczak, Katarzyna; Jiang, Xiaoyu; Jones, Michelle; Watt, James; Helfman, David M.; Moore, Jeffrey R.; Szczesna-Cordary, Danuta; Lossos, Izidore S.

    2011-01-01

    HGAL is a germinal center (GC)-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classicHodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on the actomyosin ATPase activity and on the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of GC-specific HGAL protein on lymphocyte and lymphoma cell motility. PMID:21447067

  3. Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

    PubMed

    Konno, K; Arai, K; Watanabe, S

    1979-12-01

    As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles. PMID:160911

  4. Physical and enzymatic properties of myosin from porcine brain.

    PubMed Central

    Hobbs, D S; Frederiksen, D W

    1980-01-01

    Porcine brain myosin is a cytoplasmic protein similar to, but distinct from, its muscle counterpart. It has a high K+-ATPase activity at high ionic strength in EDTA and a low Mg+2-ATPase activity that is activated fivefold by either porcine brain or rabbit skeletal muscle actin. The molecule consists of three classes of subunits, with molecular weights of approximately 195,000 , 19,000, and 16,000. Brain myosin contains less glutamic acid, less lysine, and more threonine, serine, proline, and tyrosine than skeletal muscle myosin. The brain myosin extinction coefficient at 278 nm is 0.810 cm2/mg. Hydrodynamic studies yield an S020,w of 4.95S, a D020,w of 1.07 x 10(-7) cm2/s for brain myosin, and indicate that the molecules aggregate at high ionic strength. The molecular weight of the molecule, as calculated from extrapolation of D020,w/S20,w to zero concentration, is 444,000. The intrinsic viscosity of brain myosin is 0.191 ml/mg. These data are consistent with a highly asymmetric molecular species. Circular dichroism spectroscopy indicates that brain myosin is 58-60% alpha-helical in the presence of Ca+2 ions, and that removal of Ca+2 causes a small change in the spectrum. PMID:6114756

  5. Cooperative actions between myosin heads bring effective functions.

    PubMed

    Esaki, Seiji; Ishii, Yoshiharu; Nishikawa, Masatoshi; Yanagida, Toshio

    2007-04-01

    A recent study with single molecule measurements has reported that muscle myosin, a molecular motor, stochastically generates multiple steps along an actin filament associated with the hydrolysis of a single ATP molecule [Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H., Yanagida, T., 2005. Mechanism of muscle contraction based on stochastic properties of single actomyosin motors observed in vitro. Biophysics 1, 1-19]. We have built a model reproducing such a stochastic movement of a myosin molecule incorporated with ATPase reaction cycles and demonstrated that the thermal fluctuation was a key for the function of myosin molecules [Esaki, S., Ishii, Y., Yanagida, T., 2003. Model describing the biased Brownian movement of myosin. Proc. Jpn. Acad. 79 (Ser B), 9-14]. The size of the displacement generated during the hydrolysis of single ATP molecules was limited within a half pitch of an actin filament when a single myosin molecules work separately. However, in muscle the size of the displacement has been reported to be greater than 60 nm [Yanagida, T., Arata, T., Oosawa, F., 1985. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle. Nature 316, 366-369; Higuchi et al., 1991]. The difference suggests cooperative action between myosin heads in muscle. Here we extended the model built for an isolated myosin head to a system in which myosin heads are aligned in muscle arrangement to understand the cooperativity between heads. The simulation showed that the rotation of the actin filament [Takezawa, Y., Sugimoto, Y., Wakabayashi, K., 1998. Extensibility of the actin and myosin filaments in various states of skeletal muscles as studied by X-ray diffraction. Adv. Exp. Med. Biol. 453, 309-317; Wakabayashi, K., Ueno, Y., Takezawa, Y., Sugimoto, Y., 2001. Muscle contraction mechanism: use of X-ray synchrotron radiation. Nat. Enc. Life Sci. 1-11] associated with the release of ATPase products and binding of ATP as well as interaction between myosin heads allowed the myosin filament to move greater than a half pitch of the actin filament while a single ATP molecule is hydrolyzed. Our model demonstrated that the movement is loosely coupled to the ATPase cycle as observed in muscle. PMID:17187925

  6. Phalloidin perturbs the interaction of human non-muscle myosin isoforms 2A and 2C1 with F-actin.

    PubMed

    Diensthuber, Ralph P; Mller, Mirco; Heissler, Sarah M; Taft, Manuel H; Chizhov, Igor; Manstein, Dietmar J

    2011-03-01

    Phalloidin and fluorescently labeled phalloidin analogs are established reagents to stabilize and mark actin filaments for the investigation of acto-myosin interactions. In the present study, we employed transient and steady-state kinetic measurements as well as in vitro motility assays to show that phalloidin perturbs the productive interaction of human non-muscle myosin-2A and -2C1 with filamentous actin. Phalloidin binding to F-actin results in faster dissociation of the complex formed with non-muscle myosin-2A and -2C1, reduced actin-activated ATP turnover, and slower velocity of actin filaments in the in vitro motility assay. In contrast, phalloidin binding to F-actin does not affect the interaction with human non-muscle myosin isoform 2B and Dictyostelium myosin-2 and myosin-5b. PMID:21295570

  7. An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

    PubMed Central

    Halstead, Miriam F.; Ajtai, Katalin; Penheiter, Alan R.; Spencer, Joshua D.; Zheng, Ye; Morrison, Emma A.; Burghardt, Thomas P.

    2007-01-01

    The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network. PMID:17704147

  8. Two ATPases

    PubMed Central

    Senior, Alan E.

    2012-01-01

    In this article, I reflect on research on two ATPases. The first is F1F0-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization. PMID:22822068

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

    PubMed Central

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

    2010-01-01

    SUMMARY Human ES cells are the pluripotent precursor of the three embryonic germ layers. Human ES cells 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 ES cells 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 ES cell 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 ES cells. 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 ES cells. PMID:20682449

  10. Pathway of the microtubule-kinesin ATPase.

    PubMed Central

    Johnson, K A; Gilbert, S P

    1995-01-01

    We have established pathway of the kinesin ATPase by direct measurement of each step in the pathway. Kinesin binds to microtubules with an 8-nm repeat and a stoichiometry of one kinesin monomer unit per tubulin dimer. Thus, the dimeric kinesin binds with both heads attached to the microtubule and on adjacent tubulin subunits. In the steady state, kinesin has a low ATPase activity that is limited by the rate of ADP release (< 0.01 s-1) in the absence of microtubules and is activated 2000-fold by the addition of microtubules to achieve a maximum rate of approximately 20 s-1. Transient-state kinetic analysis has provided direct measurement of individual steps of the reaction to define the pathway of the microtubule-kinesin ATPase. These studies establish that the rate-limiting step in the ATPase pathway is the release of the kinesin-product complex (K.ADP.P) from the microtubule following ATP hydrolysis. After phosphate release, the rebinding of kinesin-ADP to the microtubule is fast, accounting for the high activation of the ATPase at low microtubule concentration. This ATPase cycle explains the phenomenological differences between myosin and kinesin observed in motility assays. Kinesin remains associated with a microtubule through multiple rounds of hydrolysis, because it spends only a small fraction of its duty cycle in the dissociated state. The discussion of this paper will focus on the new data, their interpretation, and significance for mechanisms of force production. The ATPase coupling mechanism will be compared with dynein and myosin. PMID:7787062

  11. Myosin superfamily evolutionary history.

    PubMed

    Thompson, Reid F; Langford, George M

    2002-11-01

    The superfamily of myosin proteins found in eukaryotic cells is known to contain at least 18 different classes. Members are classified based on the phylogenetic analysis of the head domains located at the amino terminus of the polypeptide. While phylogenetic relationships provide insights into the functional relatedness of myosins within and between families, the evolutionary history of the myosin superfamily is not revealed by such studies. In order to establish the evolutionary history of the superfamily, we analyzed the representation of myosin gene families in a range of organisms covering the taxonomic spectrum. The amino acid sequences of 232 myosin heavy chains, as well as 65 organisms representing the protist, plant, and animal kingdoms, were included in this study. A phylogenetic tree of organisms was constructed based on several complementary taxonomic classification schemes. The results of the analysis support an evolutionary hypothesis in which myosins II and I evolved the earliest of all the myosin groups. Myosins V and XI evolved from a common myosin II-like ancestor, but the two families diverged to either the plant (XI) or animal (V) lineage. Class VII myosin appeared fourth among the families, and classes VI and IX appeared later during the early period of metazoan radiation. Myosins III, XV, and XVIII appeared after this group, and X appeared during the formative phases of vertebrate evolution. The remaining members of the myosin superfamily (IV, VI, XII, XIII, XIV, XVI, and XVII) are limited in distribution to one or more groups of organisms. The evolutionary data permits one to predict the likelihood that myosin genes absent from a given species are either missing (not found yet because of insufficient data) or lost due to a mutation that removed the gene from an organism's lineage. In conclusion, an analysis of the evolutionary history of the myosin superfamily suggests that early-appearing myosin families function as generalists, carrying out a number of functions in a variety of cell types, while more recently evolved myosin families function as specialists and are limited to a few organisms or a few cell types within organisms. PMID:12382324

  12. Unconventional myosins acting unconventionally

    PubMed Central

    Woolner, Sarah; Bement, William M.

    2016-01-01

    Unconventional myosins are proteins that bind actin filaments in an ATP-regulated manner. Because of their association with membranes, they have traditionally been viewed as motors that function primarily to transport membranous organelles along actin filaments. Recently, however, a wealth of roles for myosins that are not obviously related to organelle transport have been uncovered, including organization of F-actin, mitotic spindle regulation and gene transcription. Furthermore, it has also become apparent that the motor domains of different myosins vary strikingly in their biophysical attributes. We suggest that the assumption that most unconventional myosins function primarily as organelle transporters might be misguided. PMID:19406643

  13. Direct real-time detection of the structural and biochemical events in the myosin power stroke.

    PubMed

    Muretta, Joseph M; Rohde, John A; Johnsrud, Daniel O; Cornea, Sinziana; Thomas, David D

    2015-11-17

    A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)(2)FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin's power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered. PMID:26578772

  14. Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases.

    PubMed

    Winder, S J; Walsh, M P; Vasulka, C; Johnson, J D

    1993-12-01

    Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin] (MIANS.CaM) with a Kd of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of Pi/mol) bound to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca(2+)-/CaM-activated cyclic nucleotide phosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM interaction was Ca(2+)-dependent: half-maximal binding of calponin to MIANS.CaM occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow fluorescence kinetic analysis demonstrated that EGTA chelation of Ca2+ from CaM disrupted the MIANS.CaM-calponin complex at a rate of 1 s-1. Calponin bound MIANS.CaM at a rate of (6.0 +/- 1.8) x 10(6) M-1s-1, and melittin and unlabeled brain CaM both disrupted the MIANS.CaM-calponin complex at a rate of 0.3 +/- 0.1 s-1. These studies suggest that calponin binds CaM with 80-fold lower affinity than myosin light-chain kinase and that calponin associates with CaM much slower than it associates with caldesmon or myosin light-chain kinase. The physiological relevance of the CaM-calponin interaction was evaluated by analysis of the effects of Ca(2+)-CaM on (i) the interaction of calponin with actin and (ii) calponin-mediated inhibition of actin-activated myosin MgATPase activity. Ca(2+)-CaM half-maximally inhibited calponin (2 microM) binding to smooth and skeletal muscle actins (9 microM) at 5.4 and 11 microM CaM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8241189

  15. Rotary ATPases

    PubMed Central

    Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

    2013-01-01

    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

  16. 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. PMID:26586917

  17. The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II*

    PubMed Central

    Bloemink, Marieke J.; Melkani, Girish C.; Bernstein, Sanford I.; Geeves, Michael A.

    2016-01-01

    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. PMID:26586917

  18. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Lguillette, 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

  19. Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.

    PubMed

    Bird, Jonathan E; Takagi, Yasuharu; Billington, Neil; Strub, Marie-Paule; Sellers, James R; Friedman, Thomas B

    2014-08-26

    Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells. PMID:25114250

  20. Myosin-I nomenclature.

    PubMed

    Gillespie, P G; Albanesi, J P; Bahler, M; Bement, W M; Berg, J S; Burgess, D R; Burnside, B; Cheney, R E; Corey, D P; Coudrier, E; de Lanerolle, P; Hammer, J A; Hasson, T; Holt, J R; Hudspeth, A J; Ikebe, M; Kendrick-Jones, J; Korn, E D; Li, R; Mercer, J A; Milligan, R A; Mooseker, M S; Ostap, E M; Petit, C; Pollard, T D; Sellers, J R; Soldati, T; Titus, M A

    2001-11-26

    We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption. PMID:11724811

  1. Myosin heavy chain isoforms and smooth muscle function.

    PubMed

    Paul, R J; Hewett, T E; Martin, A F

    1991-01-01

    Using isoform specific antibodies we have verified the presence of two distinct muscle type myosin heavy chain isoforms in rat uterine muscle. We have shown that an endogenous protease can cleave a small 4 kDa region from the C-terminal of the SM1 isoform which generates a pSM1 species which comigrates with the SM2 isoform on low density SDS gels. While this cleavage can complicate isoform identification, more importantly, this cleavage was associated with a substantial increase in the actomyosin ATPase. Thus we have identified a domain at the C-terminal which may be involved in regulation of the ATPase activity. Interestingly, it is at this C-terminal, tail region of the smooth muscle myosin molecule where the only known isoform specific sequence differences are located. In skinned smooth muscle fibers of rat uterine muscle, we have also shown that differences in myosin heavy chain distribution, induced by beta-estradiol treatment of ovariectomized rats, are correlated with changes in unloaded shortening velocity. Thus our work suggests that the functional significance of myosin heavy chain isoforms in smooth muscle may be similar to that observed in striated muscle. PMID:1803896

  2. Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

    PubMed Central

    Caldwell, James T.; Melkani, Girish C.; Huxford, Tom; Bernstein, Sanford I.

    2011-01-01

    Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization. PMID:22178692

  3. Regulatory Light Chain Mutations Associated with Cardiomyopathy Affect Myosin Mechanics and Kinetics

    PubMed Central

    Greenberg, Michael J.; Watt, James D.; Jones, Michelle; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2009-01-01

    The myosin regulatory light chain (RLC) wraps around the alpha helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K showed reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation. PMID:18929571

  4. 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

  5. Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity.

    PubMed

    Tominaga, Motoki; Kojima, Hiroaki; Yokota, Etsuo; Orii, Hidefumi; Nakamori, Rinna; Katayama, Eisaku; Anson, Michael; Shimmen, Teruo; Oiwa, Kazuhiro

    2003-03-17

    High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces. PMID:12628919

  6. Myosin-II Negatively Regulates Minor Process Extension and the Temporal Development of Neuronal Polarity

    PubMed Central

    Kollins, K.M.; Hu, J.; Bridgman, P.C.; Huang, Yue-Quiao; Gallo, G.

    2009-01-01

    The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated “minor processes” (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC), mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis. PMID:19224562

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

    PubMed Central

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

    2015-01-01

    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. PMID:25751888

  8. Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

    PubMed

    Mueller, Matthias P; Goody, Roger S

    2016-08-01

    Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016. PMID:27018658

  9. Preliminary results of the ATPase reaction pathway of skeletal muscle myofibrils

    NASA Astrophysics Data System (ADS)

    Houadjeto, M.; Barman, T.; Travers, F.

    1991-05-01

    The ATPases of activated and relaxed rabbit psoas myofibrils were studied by the rapid flow quench method in a solvent of near physiological pH and ionic strength. Both types of myofibrils bind and hydrolyze ATP with transient kinetics very similar to those found with myosin. But the kcat of activated myofibrils was 100 that with the relaxed myofibrils. Relaxed myofibrils and myosin could not be distinguished kinetically.

  10. Ultraslow myosin molecular motors of placental contractile stem villi in humans.

    PubMed

    Lecarpentier, Yves; Claes, Victor; Lecarpentier, Edouard; Guerin, Catherine; Hébert, Jean-Louis; Arsalane, Abdelilah; Moumen, Abdelouahab; Krokidis, Xénophon; Michel, Francine; Timbely, Oumar

    2014-01-01

    Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n = 20) and electrical tetanic stimulation (n = 20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue. PMID:25268142

  11. Direct real-time detection of the structural and biochemical events in the myosin power stroke

    PubMed Central

    Muretta, Joseph M.; Rohde, John A.; Johnsrud, Daniel O.; Cornea, Sinziana; Thomas, David D.

    2015-01-01

    A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)2FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin’s power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered. PMID:26578772

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

    PubMed Central

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

    2015-01-01

    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

  13. 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. PMID:26827725

  14. The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state.

    PubMed

    Sellers, J R; Chock, P B; Adelstein, R S

    1983-12-10

    The correlation curve between phosphorylation and MgATPase activity suggests that the 20,000-dalton light chain of both heads of a smooth muscle myosin or heavy meromyosin (HMM) molecule must be phosphorylated before the MgATPase activity of either head can be activated by actin. The two heads of HMM appear to be phosphorylated randomly at equal rates, while those of myosin are phosphorylated in a negatively cooperative manner (Persechini, A., and Hartshorne, D.J. (1981) Science, 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. 91, 1809-1812). We have investigated the cause of this difference between HMM and myosin. We find that if myosin is first phosphorylated at high ionic strength (0.6 M KCl), where it is monomeric, and then assayed for MgATPase activity (in 0.05 M KCl), the data support a model where the two heads are phosphorylated randomly with equal rates (i.e. similarly to HMM). The correlation curves between MgATPase activity and dephosphorylation of fully phosphorylated myosin, both in a filamentous and monomeric state, are also best explained by a model where dephosphorylation of one head is sufficient to deactivate the entire molecule. With monomeric myosin, the dephosphorylation appears to occur randomly with equal rates, whereas with filamentous myosin the dephosphorylation appears to be negatively cooperative. The correlation between dephosphorylation of HMM and its MgATPase activity is more complex and is consistent with a positively cooperative dephosphorylation. Direct analyses of the time courses of phosphorylation of HMM and monomeric myosin show that a single exponential is sufficient to fit the data through greater than 90% of the reaction. However, when phosphorylation is carried out at low ionic strength (0.02 M KCl), where myosin is present as filaments, the time course consists of two exponential functions where the rate constant for the phosphorylation of one myosin head is 6-10 times greater than that for the other head which is located on the same molecule. This suggests that when myosin is polymerized into filaments the two previously indistinguishable heads either become nonequivalent or are subject to head-head interactions leading to a negatively cooperative phosphorylation reaction. PMID:6139378

  15. T cell receptor signaling and immunological synapse stability require myosin IIA

    PubMed Central

    Ilani, Tal; Vasiliver-Shamis, Gaia; Vardhana, Santosh; Bretscher, Anthony; Dustin, Michael L.

    2009-01-01

    Immunological synapses are initiated by signaling in discrete T cell receptor (TCR) microclusters and play an important role in T cell differentiation and effector functions. Synapse formation involves orchestrated motion of microclusters toward the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the role of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of TCR microclusters and that activated myosin IIA was recruited to the synapse. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted downstream of Lck and the synapse was destabilized. Thus, TCR signaling and subsequent immunological synapse formation are active processes dependent on myosin IIA. PMID:19349987

  16. Local heat activation of single myosins based on optical trapping of gold nanoparticles.

    PubMed

    Iwaki, Mitsuhiro; Iwane, Atsuko H; Ikezaki, Keigo; Yanagida, Toshio

    2015-04-01

    Myosin is a mechano-enzyme that hydrolyzes ATP in order to move unidirectionally along actin filaments. Here we show by single molecule imaging that myosin V motion can be activated by local heat. We constructed a dark-field microscopy that included optical tweezers to monitor 80 nm gold nanoparticles (GNP) bound to single myosin V molecules with nanometer and submillisecond accuracy. We observed 34 nm processive steps along actin filaments like those seen when using 200 nm polystyrene beads (PB) but dwell times (ATPase activity) that were 4.5 times faster. Further, by using DNA nanotechnology (DNA origami) and myosin V as a nanometric thermometer, the temperature gradient surrounding optically trapped GNP could be estimated with nanometer accuracy. We propose our single molecule measurement system should advance quantitative analysis of the thermal control of biological and artificial systems like nanoscale thermal ratchet motors. PMID:25736894

  17. Actin age orchestrates myosin-5 and myosin-6 run lengths.

    PubMed

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

    2015-08-01

    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 in which 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 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 toward the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks toward the pointed end of F-actin [5], traveling toward 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, whereas 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

  18. Mammalian myosin-18A, a highly divergent myosin.

    PubMed

    Guzik-Lendrum, Stephanie; Heissler, Sarah M; Billington, Neil; Takagi, Yasuharu; Yang, Yi; Knight, Peter J; Homsher, Earl; Sellers, James R

    2013-03-29

    The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells. PMID:23382379

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

    PubMed

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

    2012-06-01

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

  20. The Energetics of Allosteric Regulation of ADP Release From Myosin Heads

    PubMed Central

    Jackson, Del R.; Baker, Josh E.

    2009-01-01

    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force generating and force sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, ΔΔG-D, in the standard free energy for ADP release that results from the work, Δμmech, performed by that myosin head upon ADP release, or ΔΔG-D = Δμmech. We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires ∼ kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only ∼0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights into muscle and myosin V function. PMID:19506755

  1. Functional diversity among a family of human skeletal muscle myosin motors

    PubMed Central

    Resnicow, Daniel I.; Deacon, John C.; Warrick, Hans M.; Spudich, James A.; Leinwand, Leslie A.

    2010-01-01

    Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-IIa and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms. PMID:20080549

  2. Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

    PubMed Central

    Nag, Suman; Sommese, Ruth F.; Ujfalusi, Zoltan; Combs, Ariana; Langer, Stephen; Sutton, Shirley; Leinwand, Leslie A.; Geeves, Michael A.; Ruppel, Kathleen M.; Spudich, James A.

    2015-01-01

    Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation. PMID:26601291

  3. Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function.

    PubMed

    Nag, Suman; Sommese, Ruth F; Ujfalusi, Zoltan; Combs, Ariana; Langer, Stephen; Sutton, Shirley; Leinwand, Leslie A; Geeves, Michael A; Ruppel, Kathleen M; Spudich, James A

    2015-10-01

    Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca(2+)-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation. PMID:26601291

  4. The post-rigor structure of myosin VI and implications for the recovery stroke.

    PubMed

    Ménétrey, Julie; Llinas, Paola; Cicolari, Jérome; Squires, Gaëlle; Liu, Xiaoyan; Li, Anna; Sweeney, H Lee; Houdusse, Anne

    2008-01-01

    Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP. PMID:18046460

  5. Effect of Hindlimb Unweighting on Single Soleus Fiber Maximal Shortening Velocity and ATPase Activity

    NASA Technical Reports Server (NTRS)

    McDonald, K. S.; Fitts, R. H.

    1993-01-01

    This study characterizes the time course of change in single soleus muscle fiber size and function elicited by hindlimb un weighting (HU) and analyzes the extent to which varying durations of HU altered maximal velocity of shortening (V(sub o)), myofibrillar adenosinetriphosphatase (ATPase), and relative content of slow and fast myosin in individual soleus fibers. After 1, 2, or 3 weeks of HU, soleus muscle bundles were prepared and stored in skinning solution at -20 C. Single fibers were isolated and mounted between a motor arm and a transducer, and fiber force, V(sub o), and ATPase activity were measured. Fiber myosin content was determined by one-dimensional sodium dodecyl sulfate- (SDS) polyacrylamide gel electrophoresis. After 1, 2, and 3 weeks of HU, soleus fibers exhibited a progressive reduction in fiber diameter (16, 22, and 42%, respectively) and peak force (42, 48, and 7%, respectively). Peak specific tension was significantly reduced after 1 week of HU (18%) and showed no further change in 2-3 weeks of HU. During 1 and 3 wk of HU, fiber V(sub o) and ATPase showed a significant increase. By 3 week, V(sub o) had increased from 1.32 +/- 0.04 to 2.94 +/- 0.17 fiber lengths/s and fiber ATPase from 291 +/- 16 to 1064 +/- 128 micro-M min(sub -1) mm(sub -3). The percent fibers expressing fast myosin heavy chain increased from 4% to 29% by 3 week of HU, and V(sub o) and ATPase activity within a fiber were highly correlated. However, a large population of fibers after 1, 2, and 3 weeks of HU showed increases in V(sub o) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers. The mechanism eliciting increased fiber V(sub o) and ATPase activity was not obvious but may have been due to increases in fast myosin that went undetected on SDS gels and/or other factors unrelated to the myosin filament.

  6. A dithio-coupled kinase and ATPase assay.

    PubMed

    Chiku, Taurai; Pullela, Phani Kumar; Sem, Daniel S

    2006-10-01

    Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPbetaS (sulfur on the beta-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPbetaS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPbetaS and not with ATPbetaS. Thiol detection was done using both Ellman's reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPbetaS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z' value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening. PMID:16943391

  7. A comparison of rat myosin from fast and slow skeletal muscle and the effect of disuse

    NASA Technical Reports Server (NTRS)

    Unsworth, B. R.; Witzmann, F. A.; Fitts, R. H.

    1981-01-01

    Certain enzymatic and structural features of myosin, purified from rat skeletal muscles representative of the fast twitch glycolytic (type IIb), the fast twitch oxidative (type IIa), and the slow twitch oxidative (type I) fiber, were determined and the results were compared with the measured contractile properties. Good correlation was found between the shortening velocities and Ca(2+)-activated ATPase activity for each fiber type. Short term hind limb immobilization caused prolongation of contraction time and one-half relaxation time in the fast twitch muscles and a reduction of these contractile properties in slow twitch soleus. Furthermore, the increased maximum shortening velocity in the immobilized soleus could be correlated with increased Ca(2+)-ATPase, but no change was observed in the enzymatic activity of the fast twitch muscles. No alteration in light chain distribution with disuse was observed in any of the fiber types. The myosin from slow twitch soleus could be distinguished from fast twitch myosins on the basis of the pattern of peptides generated by proteolysis of the heavy chains. Six weeks of hind limb immobilization resulted in both an increased ATPase activity and an altered heavy chain primary structure in the slow twitch soleus muscle.

  8. Structural basis of the relaxed state of a Ca2+-regulated myosin filament and its evolutionary implications

    PubMed Central

    Woodhead, John L.; Zhao, Fa-Qing; Craig, Roger

    2013-01-01

    Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca2+ binding to the essential light chains, contributing to onoff switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca2+ binding. Cryo-electron microscopy and single-particle image reconstruction of Ca2+-regulated (scallop) filaments reveals a helical array of myosin head-pair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulationinvolving phosphorylation or Ca2+ bindingshare a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca2+-binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca2+-regulated myosin. PMID:23650385

  9. [Structural and functional bases of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of myometrium].

    PubMed

    Labyntseva, R D; Bevza, A A; Bevza, O V; Cherenok, S O; Kal'chenko, V I; Kosterin, S O

    2012-01-01

    Calix[4]arene C-97 (code is shown) is the macrocyclic compound which has lipophilic intramolecular higly-structured cavity formed by four aromatic cycles, one of which on the upper rim is modified by methylene bisphosphonic group. It was shown that calix[4]arene C-97 (100 microM) efficiently inhibits ATPase activity of myosin subfragment-1 from pig myometrium, the inhibition coefficient I(0.5) being 83 +/- 7 microM. At the same time, this compound at 100 microM concentration significantly increases the effective hydrodynamic diameter of myosin subfragment-1, that may be indicative of intermolecular complexation between the calix[4]arene and myosin head. Computer simulation methods (docking, molecular dynamics, involving the Grid) have been used to clarify structural basis of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of the myometrium; participation of hydrophobic, electrostatic and pi-pi (stacking) interactions between calix[4]arene C-97 and amino acid residues of myosin subfragment-1, some of them being located near the active site of the ATPase has been found out. PMID:22679756

  10. The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects.

    PubMed

    Walklate, Jonathan; Vera, Carlos; Bloemink, Marieke J; Geeves, Michael A; Leinwand, Leslie

    2016-05-01

    The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5-9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2-35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function. PMID:26945064

  11. Histochemical and myosin composition of vampire bat (Desmodus rotundus) pectoralis muscle targets a unique locomotory niche.

    PubMed

    Hermanson, J W; Cobb, M A; Schutt, W A; Muradali, F; Ryan, J M

    1993-09-01

    The vampire bat pectoralis muscle contains at least four fiber types distributed in a nonhomogeneous pattern. One of these fiber types, here termed IIe, can be elucidated only by adenosine triphosphatase (ATPase) histochemistry combined with reactions against antifast and antislow myosin antibodies. The histochemical and immunohistochemical observations indicate a well-developed specialization of function within specific regions of the muscle. In parallel, analyses of native myosin isoforms and myosin heavy chain isoforms indicate two points. First, the histochemical "type IIe" fiber is predominant in cranial portions of the muscle, and myosin extracted from these regions exhibits a unique electrophoretic mobility not observed in the myosin isoforms of more traditional laboratory mammals. Second, the type I fibers are confined to the pectoralis abdominalis muscle and a small adjacent region of the caudal part of the pectoralis. This pattern of type I fiber distribution is considered a derived character state compared to muscle histochemical phenotype and isoform composition in the pectoralis muscles of other phyllostomids we have studied (Artibeus jamaicensis, Artibeus lituratus, Carollia perspicillata). We relate this to the unique locomotory needs of the common vampire bat, Desmodus rotundus. PMID:8230235

  12. The Most Prevalent Freeman-Sheldon Syndrome Mutations in the Embryonic Myosin Motor Share Functional Defects*

    PubMed Central

    Walklate, Jonathan; Vera, Carlos; Bloemink, Marieke J.; Geeves, Michael A.; Leinwand, Leslie

    2016-01-01

    The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5–9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2–35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function. PMID:26945064

  13. A 170 kDa polypeptide from mung bean shares multiple epitopes with rabbit skeletal myosin and binds ADP-agarose.

    PubMed

    Qiao, L; Jablonsky, P P; Elliott, J; Williamson, R E

    1994-11-01

    A 170 kDa polypeptide that has been partially purified from mung beans is retained by ADP-agarose even in the absence of divalent cations when most non-myosin ATPases and kinases do not bind. Attempts to demonstrate a myosin-like ATPase activity were inconclusive, however, and the protein accounts at most for only a small part of the total K+ EDTA ATPase activity of mung bean extracts. All four monoclonal antibodies raised to the 170 kDa polypeptide react with rabbit skeletal muscle myosin and localize the 170 kDa polypeptide in mung bean root tip cells to the actin-containing phragmoplast and to sites dispersed throughout the cytoplasm which probably include some but not all actin cables. These 4 monoclonals and 3 commercially available antimyosin monoclonals all recognise rabbit skeletal myosin and 160-170 kDa proteins that are present in two other angiosperms tested. In addition, a 158 kDa protein of mung bean reacts with only one antibody and does not bind ADP-agarose. We conclude that strong but not yet conclusive evidence points to the 160-170 kDa proteins of angiosperms being a widely conserved form of myosin heavy chain. PMID:7534549

  14. Myosinome: a database of myosins from select eukaryotic genomes to facilitate analysis of sequence-structure-function relationships.

    PubMed

    Syamaladevi, Divya P; Sunitha, Margaret S; Kalaimathy, S; Reddy, Chandrashekar C; Iftekhar, Mohammed; Pasha, Shaik N; Sowdhamini, R

    2012-01-01

    Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms (Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome. PMID:23189029

  15. Role of the Essential Light Chain in the Activation of Smooth Muscle Myosin by Regulatory Light Chain Phosphorylation

    PubMed Central

    Taylor, Kenneth A.; Feig, Michael; Brooks, Charles L.; Fagnant, Patricia M.; Lowey, Susan; Trybus, Kathleen M.

    2014-01-01

    The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin. PMID:24361582

  16. 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

  17. ATPase-coupled release control from polyion complex capsules encapsulating muscle proteins.

    PubMed

    Sugiura, Kousuke; Ohkawa, Kousaku; Hirai, Toshihiro; Fujii, Toshihiro

    2007-04-10

    In the present study, a muscle contractile protein complex, actomyosin, has been successfully encapsulated into gellan-chitosan polyion complex (PIC) capsules. The recovery of the myosin-ATPase activity is approximately 50% and the Mg2+-ATPase activity is stimulated by the presence of F-actin, which implies the formation of the actomyosin complex inside the capsule. Furthermore, encapsulation could protect the myosin, F-actin, and actomyosin inside from hydrolysis by proteases. Two small proteins, myoglobin and cytochrome c, have been used in the release tests. The release of myoglobin is not affected by the ionic strength of the external solution, while the release of cytochrome c increases with increasing ionic strength. The maximal releases are found in the external pH solution close to the isoelectric points of each protein. The Mg2+-ATP complex itself reduces the release percentages of the small proteins from the PIC capsule. The release amounts further decrease when coexisting with Mg2+-ATP and the encapsulated actomyosin, which indicates the release regulation by actomyosin. The present study suggests that the ATPase-coupled sliding motion of the myosin-F-actin filaments modifies the pore size of the polymer networks in the PIC capsule membranes. PMID:17429832

  18. Direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin V.

    PubMed

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

    2015-11-24

    Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor-acceptor pairs to examine the kinetics of lever arm swing during the recovery and power stroke phases of the ATPase cycle. During the recovery stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the power stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-Å change in distance between the pre- and postpower stroke states and shows that the lever arm is more dynamic in the postpower stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid power stroke that gates the release of phosphate, whereas a second slower power stroke may be important for mediating strain sensitivity. PMID:26553992

  19. 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

  20. Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle.

    PubMed

    Jiang, H; Rao, K; Halayko, A J; Liu, X; Stephens, N L

    1992-12-01

    Previous studies have identified changes of mechanical properties of airway smooth muscle (ASM) from a canine model of atopic airway hyperreactivity. These changes, including increased maximum shortening capacity (delta Lmax) and early shortening velocity (Vo), may be responsible for the airway hyperresponsiveness in asthma. We have suggested that these changes may be due to increased actomyosin ATPase activity, controlled via phosphorylation of the 20 kD myosin light chain (MLC20) by MLC kinase (MLCK). Therefore, ATPase activity, MLC20 phosphorylation, and MLCK content and activity were assessed in tracheal and bronchial smooth muscles (TSM and BSM) of ragweed pollen-sensitized dogs (S) and their littermate controls (C). Specific ATPase activities from STSM and SBSM were significantly higher than their control counterparts (CTSM, CBSM). Phosphorylation of MLC20 in STSM was greater both at rest and during electrical stimulation due to the increased amount of MLCK in STSM and SBSM by 30 and 25%, respectively. MLCK activity was also increased significantly in STSM and SBSM (from 46.99 +/- 8.33 and 42.85 +/- 5.92 to 91.9 +/- 6.43 and 64.12 +/- 7.88 32P mmol/mg fresh tissue weight/min respectively [mean +/- SEM]). When normalized to the amount of MLCK in the tissue, however, specific MLCK activity in STSM and SBSM was similar to that in controls. It is unlikely that myosin phosphatase plays any role in the changes of MLC20 phosphorylation in sensitized animals. Peptide mapping showed no visible change in primary structure of MLCK in STSM and SBSM compared with those of controls. We report that ASM actomyosin ATPase activity is increased in STSM and SBSM. The increased ATPase activity is the result of increased MLC20 phosphorylation, the latter likely resulting from the increased MLCK content, which may account for the hyperresponsiveness found in ASM from these animals. PMID:1449804

  1. 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

  2. Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

    PubMed

    Sugi, Haruo; Chaen, Shigeru; Akimoto, Tsuyoshi; Minoda, Hiroki; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Sugiura, Seiryo

    2015-01-01

    Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed. PMID:26498981

  3. Electron microscopic recording of myosin head power stroke in hydrated myosin filaments

    PubMed Central

    Sugi, Haruo; Chaen, Shigeru; Akimoto, Tsuyoshi; Minoda, Hiroki; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Sugiura, Seiryo

    2015-01-01

    Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed. PMID:26498981

  4. Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila

    PubMed Central

    Jordan, Pascale; Karess, Roger

    1997-01-01

    The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20. PMID:9412474

  5. Mn2+Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance

    PubMed Central

    Astashkin, Andrei V.; Nesmelov, Yuri E.

    2013-01-01

    Pulsed electron paramagnetic resonance at the microwave Ka band (~30 GHz) was used to study the coordination of adenosine nucleotides to Mn2+ at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electronnuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mnnucleotide complexes. Therefore, to directly detect binding of the Mnnucleotide to myosin, we used nonhydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electronelectron double resonance to detect spin probemanganese dipolar interaction. We found that under substoichiometric conditions, both MnAMPPNP and MnADPAlF4 form a complex with myosin, and MnADP does not form such a complex. This correlates well with the biological dissociation of MgADP from myosin after the hydrolysis of ATP. The analysis of 31P ENDOR spectra reveals that in MnAMPPNP, MnATP, and MnADP at myosin or in solution, the nucleotide is coordinated to Mn2+ by two phosphate groups, whereas in MnADPAlF4, only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of MnADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ions is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metalprotein binding when such binding does not provide reliable spectroscopic signatures. PMID:23121488

  6. 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 different from strong binding attachments.

  7. Myosin V from Drosophila reveals diversity of motor mechanisms within the myosin V family.

    PubMed

    Tóth, Judit; Kovács, Mihály; Wang, Fei; Nyitray, László; Sellers, James R

    2005-08-26

    Myosin V is the best characterized vesicle transporter in vertebrates, but it has been unknown as to whether all members of the myosin V family share a common, evolutionarily conserved mechanism of action. Here we show that myosin V from Drosophila has a strikingly different motor mechanism from that of vertebrate myosin Va, and it is a nonprocessive, ensemble motor. Our steady-state and transient kinetic measurements on single-headed constructs reveal that a single Drosophila myosin V molecule spends most of its mechanochemical cycle time detached from actin, therefore it has to function in processive units that comprise several molecules. Accordingly, in in vitro motility assays, double-headed Drosophila myosin V requires high surface concentrations to exhibit a continuous translocation of actin filaments. Our comparison between vertebrate and fly myosin V demonstrates that the well preserved function of myosin V motors in cytoplasmic transport can be accomplished by markedly different underlying mechanisms. PMID:15980429

  8. Sliding distance of actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle

    NASA Astrophysics Data System (ADS)

    Yanagida, Toshio; Arata, Toshiaki; Oosawa, Fumio

    1985-07-01

    Muscle contraction results from a sliding movement of actin filaments induced by myosin crossbridges on hydrolysis of ATP1,2, and many non-muscle cells are thought to move using a similar mechanism3-5. The molecular mechanism of muscle contraction, however, is not completely understood6,7. One of the major problems is the mechanochemical coupling at high velocity under near-zero load8-13. Here, we report measurements of the sliding distance of an actin filament induced by a myosin crossbridge during one ATP hydrolysis cycle in an unloaded condition. We used single sarcomeres from which the Z-lines, structures which anchor the thin filaments in the sarcomere, had been completely removed by calcium-activated neutral protease (CANP)14 and trypsin, and measured both the sliding velocity of single actin filaments along myosin filaments and the ATPase activity during sliding. Our results show that the average sliding distance of the actin filament is >=600 Å during one ATP cycle, much longer than the length of power stroke of myosin crossbridges deduced from mechanical studies of muscle, which is of the order of 80 Å (for example, ref. 15).

  9. Comparison of the variable loop regions of myosin heavy chain genes from Antarctic and temperate isopods.

    PubMed

    Holmes, J M; Whiteley, N M; Magnay, J L; El Haj, A J

    2002-03-01

    The evolutionary adaptations of functional genes to life at low temperatures are not well characterised in marine and fresh water invertebrates. Temperature has been shown to affect the functional characteristics of fish muscles, with changes in the velocity of shortening and ATPase activity being associated with myosin heavy chain (MyHC) isoform composition and the structure of the surface loop regions. Two PCR products spanning loops 1 and 2 of a MyHC gene from an Antarctic isopod (Glyptonotus antarcticus) were sequenced and compared with those of a temperate isopod (Idotea resecata), slow and fast fibres from lobster (Homarus gammarus) and a cold water amphipod (Eulimnogammarus verrucosus), revealing specific differences between the species, possibly related to fibre type and habitat temperature. The loop 2 region from G. antarcticus myosin was cloned and used for Northern analysis of total RNA from the other species. The cloned myosin cDNA hybridised specifically to a 6.6-kb transcript, in G. antarcticus muscle. In contrast, cDNA probes for lobster slow myosin and actin hybridised to muscle RNA from all species, demonstrating that a distinct MyHC isoform is expressed in the Antarctic isopod, as opposed to the temperate species. The inter- and intra-specific sequence differences in loop 2 region suggest that this may be a site for muscle adaptation to enable function at the low temperatures found in the Southern Ocean. PMID:11959017

  10. 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.

  11. Myosin II regulates actin rearrangement-related structural synaptic plasticity during conditioned taste aversion memory extinction.

    PubMed

    Bi, Ai-Ling; Wang, Yue; Zhang, Shuang; Li, Bo-Qin; Sun, Zong-Peng; Bi, Hong-Sheng; Chen, Zhe-Yu

    2015-03-01

    Similar to memory formation, memory extinction is also a new learning process that requires synaptic plasticity. Actin rearrangement is fundamental for synaptic plasticity, however, whether actin rearrangement in the infralimbic cortex (IL) plays a role in memory extinction, as well as the mechanisms underlying it, remains unclear. Here, using a conditioned taste aversion (CTA) paradigm, we demonstrated increased synaptic density and actin rearrangement in the IL during the extinction of CTA. Targeted infusion of an actin rearrangement inhibitor, cytochalasin D, into the IL impaired memory extinction and de novo synapse formation. Notably, we also found increased myosin II phosphorylation in the IL during the extinction of CTA. Microinfusion of a specific inhibitor of the myosin II ATPase, blebbistatin (Blebb), into the IL impaired memory extinction as well as the related actin rearrangement and changes in synaptic density. Moreover, the extinction deficit and the reduction of synaptic density induced by Blebb could be rescued by the actin polymerization stabilizer jasplakinolide (Jasp), suggesting that myosin II acts via actin filament polymerization to stabilize synaptic plasticity during the extinction of CTA. Taken together, we conclude that myosin II may regulate the plasticity of actin-related synaptic structure during memory extinction. Our studies provide a molecular mechanism for understanding the plasticity of actin rearrangement-associated synaptic structure during memory extinction. PMID:24337340

  12. Dynamics of myosin replacement in skeletal muscle cells.

    PubMed

    Ojima, Koichi; Ichimura, Emi; Yasukawa, Yuya; Wakamatsu, Jun-Ichi; Nishimura, Takanori

    2015-11-15

    Highly organized thick filaments in skeletal muscle cells are formed from ~300 myosin molecules. Each thick-filament-associated myosin molecule is thought to be constantly exchanged. However, the mechanism of myosin replacement remains unclear, as does the source of myosin for substitution. Here, we investigated the dynamics of myosin exchange in the myofibrils of cultured myotubes by fluorescent recovery after photobleaching and found that myofibrillar myosin is actively replaced with an exchange half-life of ~3 h. Myosin replacement was not disrupted by the absence of the microtubule system or by actomyosin interactions, suggesting that known cytoskeletal systems are dispensable for myosin substitution. Intriguingly, myosin replacement was independent of myosin binding protein C, which links myosin molecules together to form thick filaments. This implies that an individual myosin molecule rather than a thick filament functions as an exchange unit. Furthermore, the myosin substitution rate was decreased by the inhibition of protein synthesis, suggesting that newly synthesized myosin, as well as preexisting cytosolic myosin, contributes to myosin replacement in myofibrils. Notably, incorporation and release of myosin occurred simultaneously in myofibrils, but rapid myosin release from myofibrils was observed without protein synthesis. Collectively, our results indicate that myosin shuttles between myofibrils and the nonmyofibrillar cytosol to maintain a dynamic equilibrium in skeletal muscle cells. PMID:26377314

  13. 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

  14. 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

  15. Effect of trimetazidine and verapamil on the cardiomyopathic hamster myosin phenotype

    PubMed Central

    D'hahan, Nathalie; Taouil, Karima; Janmot, Chantal; Morel, Jean-Emile

    1998-01-01

    In this study we investigated whether long-term trimetazidine (anti-ischaemic drug) therapy alters the ventricular myosin heavy chain (MHC) isoform composition in a model of cardiomyopathy. MHC isoforms were analysed in the native state by electrophoresis in a pyrophosphate buffer. Myosin isoform patterns were studied in cardiac muscle from cardiomyopathic hamsters (CMH) of the BIO 14 : 6 strain during the time course of the disease and compared with those of healthy golden hamsters (F1B). The correlation between myosin profile and Ca2+-activated ATPase activity was determined from 220 days. At the stage of insufficiency (350 days), CMH presented the most abnormal phenotype with 53% V1-24% V3 compared to 79% V1-7% V3 (P<0.001), in F1B. Trimetazidine was administered to cardiomyopathic hamsters from the early stage of active disease (30 days) to the congestive stages (220–350 days). Within 65 days, trimetazidine treatment, in CMH and F1B, reduced V1 to a low level (53% and 62%, respectively), which remained constant throughout the treatment. This level was similar to that in 220 and 350 days-old untreated-CMH. In sharp contrast, a standard calcium blocker, verapamil, administered to CMH in the same conditions resulted in a higher V1 (about 70%) and higher global myosin ATPase activity from 220 days. Previous results in terms of hypertrophy and survival, compared to these results, suggest that verapamil and trimetazidine treatments reveal a disssociation between ventricular hypertrophy and isomyosin distribution. In addition, the shift in favour of V3 may not necessarily be an aggravating factor of the disease but an adaptative compensatory event. PMID:9517378

  16. 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.

  17. Nuclear actin and myosins in adenovirus infection.

    PubMed

    Fuchsova, Beata; Serebryannyy, Leonid A; de Lanerolle, Primal

    2015-11-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  18. Coordinate changes in Myosin heavy chain isoform gene expression are selectively associated with alterations in dilated cardiomyopathy phenotype.

    PubMed Central

    Abraham, W. T.; Gilbert, E. M.; Lowes, B. D.; Minobe, W. A.; Larrabee, P.; Roden, R. L.; Dutcher, D.; Sederberg, J.; Lindenfeld, J. A.; Wolfel, E. E.; Shakar, S. F.; Ferguson, D.; Volkman, K.; Linseman, J. V.; Quaife, R. A.; Robertson, A. D.; Bristow, M. R.

    2002-01-01

    BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM. PMID:12520092

  19. Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

    PubMed Central

    Murata, K; Hirano, K; Villa-Moruzzi, E; Hartshorne, D J; Brautigan, D L

    1997-01-01

    Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration. Images PMID:9247646

  20. 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...

  1. 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

  2. Functional studies of individual myosin molecules.

    PubMed

    Dantzig, Jody A; Liu, Tim Y; Goldman, Yale E

    2006-10-01

    The "conventional" isoform of myosin that polymerizes into filaments (myosin II) is the molecular motor powering contraction in all three types of muscle. Considerable attention has been paid to the developmental progression, isoform distribution, and mutations that affect myocardial development, function, and adaptation. Optical trap (laser tweezer) experiments and various types of high-resolution fluorescence microscopy, capable of interrogating individual protein motors, are revealing novel and detailed information about their functionally relevant nanometer motions and pico-Newton forces. Single-molecule laser tweezer studies of cardiac myosin isoforms and their mutants have helped to elucidate the pathogenesis of familial hypertrophic cardiomyopathies. Surprisingly, some disease mutations seem to enhance myosin function. More broadly, the myosin superfamily includes more than 20 nonfilamentous members with myriad cellular functions, including targeted organelle transport, endocytosis, chemotaxis, cytokinesis, modulation of sensory systems, and signal transduction. Widely varying genetic, developmental and functional disorders of the nervous, pigmentation, and immune systems have been described in accordance with these many roles. Compared to the collective nature of myosin II, some myosin family members operate with only a few partners or even alone. Individual myosin V and VI molecules can carry cellular vesicular cargoes much farther distances than their own size. Laser tweezer mechanics, single-molecule fluorescence polarization, and imaging with nanometer precision have elucidated the very different mechano-chemical properties of these isoforms. Critical contributions of nonsarcomeric myosins to myocardial development and adaptation are likely to be discovered in future studies, so these techniques and concepts may become important in cardiovascular research. PMID:17132771

  3. Rotational dynamics of actin-bound myosin heads in active myofibrils.

    PubMed

    Berger, C L; Thomas, D D

    1993-04-13

    We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo. PMID:8385491

  4. Coupling of Two Non-processive Myosin 5c Dimers Enables Processive Stepping along Actin Filaments

    PubMed Central

    Gunther, Laura K.; Furuta, Ken'ya; Bao, Jianjun; Urbanowski, Monica K.; Kojima, Hiroaki; White, Howard D.; Sakamoto, Takeshi

    2014-01-01

    Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 3036?nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells. PMID:24809456

  5. Cold-sensitive mutations of Dictyostelium myosin heavy chain highlight functional domains of the myosin motor

    SciTech Connect

    Patterson, B.; Spudich, J.A.

    1996-06-01

    Dictyostelium provides a powerful environment for characterization of myosin II function. It provides well-established biochemical methods for in vitro analysis of myosin`s properties as well as an array of molecular genetic tools. The absence of myosin function results in an array of phenotypes that can be used to genetically manipulate myosin function. We have previously reported methods for the isolation and identification of rapid-effect cold-sensitive myosin II mutations in Dictyostelium. Here, we report the development and utilization of a rapid method for localizing these point mutations. We have also sequenced 19 mutants. The mutations show distinct clustering with respect to three-dimensional location and biochemically characterized functional domains of the protein. We conclude that these mutants represent powerful tools for understanding the mechanisms driving this protein motor. 36 refs., 5 figs., 2 tabs.

  6. 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.

  7. Myosin IIC: A Third Molecular Motor Driving Neuronal Dynamics

    PubMed Central

    Wylie, Steven R.

    2008-01-01

    Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells. PMID:18614800

  8. Differential localization of unconventional myosin I and nonmuscle myosin II during B cell spreading.

    PubMed

    Sumoza-Toledo, Adriana; Gillespie, Peter G; Romero-Ramirez, Hector; Ferreira-Ishikawa, Hellen C; Larson, Roy E; Santos-Argumedo, Leopoldo

    2006-10-15

    Cross-linking of CD44 in vitro promotes chemokinesis and actin-based dendrite formation in T and B cells. However, the mechanisms by which the adhesion molecule CD44 induces cytoskeleton activation in lymphocytes are still poorly understood. In this study, we have investigated whether myosin isoforms are involved in CD44-dependent dendrite formation in activated B cells. Pharmacological inhibition of myosin with 2,3-butanedione monoxime strongly affected spreading and dendrite formation, suggesting that these cellular motors may participate in these phenomena. Furthermore, immunofluorescence analysis showed differences in subcellular localization of class I and class II myosin during B cell spreading. In response to CD44 cross-linking, myosin-1c was polarized to lamellipodia, where F-actin was high. In contrast, the distribution of cytosplasmic nonmuscle class II myosin was not altered. Expressions of myosin-1c and II were also demonstrated in B cells by Western blot. Although the inhibition of PLCgamma, PI3K and MEK-1 activation affected the spreading and dendrite formation in activated B cells, only PLCgamma and MEK-1 inhibition correlated with absence of myosin-1c polarization. Additionally, myosin-1c polarization was observed upon cross-linking of other surface molecules, suggesting a common mechanism for B cell spreading. This work shows that class I and class II myosin are expressed in B cells, are differentially distributed, and may participate in the morphological changes of these cells. PMID:16919270

  9. Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

    PubMed Central

    Markov, Denis I.; Zubov, Eugene O.; Nikolaeva, Olga P.; Kurganov, Boris I.; Levitsky, Dmitrii I.

    2010-01-01

    We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain. PMID:21151434

  10. Homology model of nonmuscle myosin heavy chain IIA and binding mode analysis with its inhibitor blebbistatin.

    PubMed

    Lv, Yanni; Lu, Shuai; Lu, Tao; Kou, Junping; Yu, Boyang

    2013-04-01

    Nonmuscle myosin heavy chain IIA (NMMHC IIA, gene code: MYH9) plays a critical role in physiological and pathological functions. A homology model of NMMHC IIA was constructed based on the crystal structure of smooth muscle myosin II. Blebbistatin, a myosin II ATPase inhibitor, had been found to bind to NMMHC IIA with Leu228 as the important amino acid residue and van der Waals contacts as the main force of the interaction. The final complex demonstrated that the destruction of the salt bridge occurred between the Arg204 and Glu427 residues when blebbistatin was present. Molecular dynamic simulation of the complex showed that the binding affinity of blebbistatin to NMMHC IIA was strongly sensitive to the nucleotide binding region and actin binding region. The disturbance of the two regions increased the enhancement of the binding cavity with blebbistatin and resulted in a slightly more expanded conformation in the nucleotide binding region and actin binding region. A combined pharmacophore- and docking-based virtual screening was performed to identify several saponins as potential inhibitors for NMMHC IIA. These findings introduce new insights on the binding mode of blebbistatin and NMMHC IIA and novel leading compounds from natural products for NMMHC IIA-related diseases. PMID:23315199

  11. Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages

    SciTech Connect

    Mykles, D.L.

    1985-01-01

    Fast and slow muscles from the claws and abdomen of the American lobster Homarus americanus were examined for adenosine triphosphatase (ATPase) activity and for differences in myofibrillar proteins. Both myosin and actomyosin ATPase were correlated with fiber composition and contractile speed. Four distinct patterns of myofibrilla proteins observed in sodium dodecyl sulfate-polyacrylamide gels were distinguished by different assemblages of regulatory and contractile protein variants. A total of three species of troponin-T, five species of troponin-I, and three species of troponin-C were observed. Lobster myosins contained two groups of light chains (LC), termed alpha and beta. There were three ..cap alpha..-LC variants and two ..beta..-LC variants. There were no apparent differences in myosin heavy chain, actin, and tropomyosin. Only paramyosin showed a pattern completely consistent with muscle fiber type: slow fibers contained a species (105 kD) slightly smaller than the principle variant (110 kD) in fast fibers. It is proposed that the type of paramyosin present could provide a biochemical marker to identify the fiber composition of muscles that have not been fully characterized. The diversity of troponin and myosin LC variants suggests that subtle differences in physiological performance exist within the broader categories of fast- and slow-twitch muscles. 31 references, 6 figures, 2 tables.

  12. Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish.

    PubMed

    Abrams, Joshua; Einhorn, Zev; Seiler, Christoph; Zong, Alan B; Sweeney, H Lee; Pack, Michael

    2016-05-01

    Smooth muscle contraction is controlled by the regulated activity of the myosin heavy chain ATPase (Myh11). Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt), which converts a highly conserved tryptophan to arginine (W512R) in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here, we report two newly identified missense mutations in the switch-1 (S237Y) and coil-coiled (L1287M) domains of Myh11 that fail to complement mlt Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell-invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which, in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter the regulation of smooth muscle myosin might be risk factors for diseases affecting the intestine, vasculature, and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress. PMID:26893369

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

    PubMed

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

    2016-04-15

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

  15. Sliding distance per ATP molecule hydrolyzed by myosin heads during isotonic shortening of skinned muscle fibers.

    PubMed Central

    Higuchi, H; Goldman, Y E

    1995-01-01

    We measured isotonic sliding distance of single skinned fibers from rabbit psoas muscle when known and limited amounts of ATP were made available to the contractile apparatus. The fibers were immersed in paraffin oil at 20 degrees C, and laser pulse photolysis of caged ATP within the fiber initiated the contraction. The amount of ATP released was measured by photolyzing 3H-ATP within fibers, separating the reaction products by high-pressure liquid chromatography, and then counting the effluent peaks by liquid scintillation. The fiber stiffness was monitored to estimate the proportion of thick and thin filament sites interacting during filament sliding. The interaction distance, Di, defined as the sliding distance while a myosin head interacts with actin in the thin filament per ATP molecule hydrolyzed, was estimated from the shortening distance, the number of ATP molecules hydrolyzed by the myosin heads, and the stiffness. Di increased from 11 to 60 nm as the isotonic tension was reduced from 80% to 6% of the isometric tension. Velocity and Di increased with the concentration of ATP available. As isotonic load was increased, the interaction distance decreased linearly with decrease of the shortening velocity and extrapolated to 8 nm at zero velocity. Extrapolation of the relationship between Di and velocity to saturating ATP concentration suggests that Di reaches 100-190 nm at high shortening velocity. The interaction distance corresponds to the sliding distance while cross-bridges are producing positive (working) force plus the distance while they are dragging (producing negative forces). The results indicate that the working and drag distances increase as the velocity increases. Because Di is larger than the size of either the myosin head or the actin monomer, the results suggest that for each ATPase cycle, a myosin head interacts mechanically with several actin monomers either while working or while producing drag. PMID:8534820

  16. Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

    PubMed Central

    Xu, Li; Bretscher, Anthony

    2014-01-01

    Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

  17. 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.

  18. The 3-(bromoacetamido)-propylamine hydrochloride: A novel sulfhydryl reagent and its future potential in the configurational study of S1-myosin

    NASA Technical Reports Server (NTRS)

    Sharma, Prasanta; Cheung, Herbert C.

    1989-01-01

    Configurational study of S1-Myosin is an important step towards understanding force generation in muscle contraction. Previously reported NMR studies were corroborated. A new compound was synthesized, 3-(Bromoacetamido)-propylamine hydrochloride. Its potential as a sulfhydryl reagent provides an indirect but elegant approach towards future structural elucidation of S1-Myosin. The preliminary investigation has shown that this compound, BAAP, reacted with S1 in the absence of MgADP. The modified enzyme had a 2-fold increase in CaATPase activity and no detectable K-EDTA ATPase activity. Reaction of BAAP with S1 in the presence of MgADP resulted in a modified enzyme which retained a Ca-ATPase activity that was about 60 percent of the unmodified S1 and had essentially zero K-EDTA ATPase activity. Sulfhydryl titration indicated that about 1.5 and 3.5 SH groups per S1 molecule were blocked by BAAP in the absence and presence of MgADP, respectively. When coupled to a carboxyl group of EDTA, the resulting reagent could become a useful SH reagent in which chelated paramagnetic or luminescent lanthanide ions can be exploited to probe S1 conformation.

  19. 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 Central

    Tanner, Bertrand C.W.; McNabb, Mark; Palmer, Bradley M.; Toth, Michael J.; Miller, Mark S.

    2014-01-01

    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 simulation 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

  20. Formation of the myosin.ADP.gallium fluoride complex and its solution structure by small-angle synchrotron X-ray scattering.

    PubMed

    Maruta, S; Uyehara, Y; Homma, K; Sugimoto, Y; Wakabayashi, K

    1999-01-01

    In the presence of MgADP, a novel phosphate analogue of gallium fluoride (GaFn) forms a ternary complex with the myosin subfragment-1 (S-1), in the same way that has been previously reported with aluminum fluoride (AlF4-), beryllium fluoride (BeFn), scandium fluoride (ScFn), and vanadate (Vi), and this complex formation may mimic different states along the ATPase kinetic pathway. This novel complex has been characterized and compared with other complexes to ascertain whether it forms a transition-state analogue of myosin ATPase. The complex formed quickly, although several times slower than the BeFn complex. The half-life of the myosin.ADP.GaFn complex was about 50 h at 4 degreesC. The formation of the myosin.ADP.GaFn complex was accompanied by an increase in tryptophane fluorescence, similar to that observed upon the addition of ATP, but slightly lower than that of the M**.ADP.Pi complex. Upon addition of GaFn to acto-myosin.ADP, acto-myosin did not dissociate, and the S-1.ADP.GaFn complex was scarcely decomposed by actin, like the AlF4- and ScFn complexes but unlike the BeFn and Vi complexes. The conformations at the localized region of SH1, SH2, and RLR, which are very accessible to the binding of ATP, were studied by fluorescent labeling and chemical modification, and the results suggested that these conformations are very similar to that of the M**.ADP.Pi state. Small-angle X-ray solution scattering showed that the radius of gyration value decreases by about 3 A when S-1 forms an S-1.ADP.GaFn complex, suggesting that the shape of the complex becomes compact or rounded in shape, similar to that in the presence of ATP or complexes with other phosphate analogues, and thus mimics the myosin**.ADP.Pi state closely. The overall results may indicate that the complex mimics a somewhat different transient state from that of other complexes but has a similar global conformation along the ATPase kinetic pathway. PMID:9880815

  1. 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.

  2. Regulation and recycling of myosin V.

    PubMed

    Taylor, Kenneth A

    2007-02-01

    Recently there has been considerable progress in our understanding of regulation for unconventional myosin-V through elucidation of the structure of its inactive conformation and the factors that affect stability of this conformation. The inactive conformation is a folded compact structure characterized by interactions between the myosin head and the C-terminal cargo binding domain. Concentrations of Ca2+ greater than 10 microM disrupt folding. The 3-D structure determined by cryoelectron tomography of 2-D arrays in one study and electron micrographs of isolated molecules reported in another reveal similar features, but suggest different F-actin affinities for the inactive conformation. This has raised the question of how inactive myosin-V is recycled to other sites for additional rounds of cargo transport. PMID:17208425

  3. 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 kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

  4. A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila.

    PubMed

    Achal, Madhulika; Trujillo, Adriana S; Melkani, Girish C; Farman, Gerrie P; Ocorr, Karen; Viswanathan, Meera C; Kaushik, Gaurav; Newhard, Christopher S; Glasheen, Bernadette M; Melkani, Anju; Suggs, Jennifer A; Moore, Jeffrey R; Swank, Douglas M; Bodmer, Rolf; Cammarato, Anthony; Bernstein, Sanford I

    2016-06-01

    An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue. PMID:27107639

  5. Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

    PubMed Central

    Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta; Moore, Jeffrey R.

    2016-01-01

    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

  6. 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.

  7. Left-right asymmetry: class I myosins show the direction.

    PubMed

    Spéder, Pauline; Noselli, Stéphane

    2007-02-01

    Myosins are actin-based molecular motors that are found in almost all eukaryotes. Phylogenetic analysis allows the discrimination of 37 different types of myosins, most with unknown functions. Recent work in Drosophila has revealed a crucial role for type ID unconventional myosin in left-right asymmetry. Mutations in Myosin ID completely reverse the left-right axis (situs inversus), a phenotype that is dependent on an intact actin cytoskeleton. How this myosin might orient the left-right axis has began to be elucidated by showing that it interacts directly with beta-catenin, suggesting that myosin ID interacts with the adherens junction to control the direction of organ looping. This is the first demonstration of a role of a myosin in body patterning. PMID:17174542

  8. Relationship of the Membrane ATPase from Halobacterium saccharovorum to Vacuolar ATPases

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Bowman, Emma J.; Hochstein, Lawrence I.

    1991-01-01

    Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan in- hibited the hatobacterial ATPase also in a nucleotide- protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuotar and the F-type ATPases.

  9. Indirect myosin immunocytochemistry for the identification of fibre types in equine skeletal muscle

    NASA Technical Reports Server (NTRS)

    Sinha, A. K.; Rose, R. J.; Pozgaj, I.; Hoh, J. F.

    1992-01-01

    The histochemical ATPase method for muscle fibre typing was first described by Brooke and Kaiser in 1970. However, problems have been found with the subdivision of type II fibres using this technique. To determine whether indirect myosin immunocytochemistry using anti-slow (5-4D), anti-fast (1A10) and anti-fast red (5-2B) monoclonal antibodies with cross reactivity for type I, II and IIa fibres, respectively, in a number of species, could identify three fibre types in equine skeletal muscle, data on fibre type composition and fibre size obtained using the two different techniques were compared. Results indicate that different myosin heavy chains can coexist in single equine muscle fibres. Type I and type II fibres were identified by immunocytochemistry, but subdivision of type II fibres was not possible. Although the percentage of type I and type II fibres was not significantly different for the two techniques, a few fibres reacted with both the 1A10 and 5-4D antibodies.

  10. Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain.

    PubMed Central

    Babij, P

    1993-01-01

    Previous work demonstrated that the rabbit smooth muscle myosin heavy chain gene showed sequence divergence at the 25kDa/50kDa junction of the S1 subfragment when compared to chicken gizzard and chicken epithelial nonmuscle myosin. RNase protection analysis with a probe spanning this region detected two partially protected fragments which were not present in RNA from vascular tissue and only found in RNA from visceral tissue. The polymerase chain reaction was used to amplify a 162bp product from primers spanning the putative region of divergence and DNA sequence analysis revealed a seven amino acid insertion not previously detected in other characterised cDNA clones. RNase protection analysis using the PCR product as probe showed that the inserted sequence was expressed exclusively in RNA from visceral tissue. Similar RNA analysis showed that the visceral isoform was not expressed in 20 day fetal rabbit smooth muscle tissues. These results indicated that the new visceral isoform was expressed in a tissue-specific and developmentally regulated manner. Genomic DNA sequencing and mapping of the exon-intron boundaries showed that the visceral isoform was the product of cassette-type alternative splicing. The inclusion of a visceral-specific sequence near the Mg-ATPase domain and at the 25kDa/50kDa junction suggests that the visceral isoform may be important for myosin function in smooth muscle cells. Images PMID:8464739

  11. Myosin VI Stabilizes an Actin Network during Drosophila Spermatid Individualization

    PubMed Central

    Lenartowska, Marta; Miller, Kathryn G.

    2006-01-01

    Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells. PMID:16571671

  12. 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.

  13. The Role of Myosin II Motor Activity in Distributing Myosin Asymmetrically and Coupling Protrusive Activity to Cell Translocation

    PubMed Central

    2006-01-01

    Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at ≥30 μM inhibited anterior redistribution of myosin IIA, with 100 μM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward. PMID:16855019

  14. Time dependent effects on contractile properties, fibre population, myosin light chains and enzymes of energy metabolism in intermittently and continuously stimulated fast twitch muscles of the rabbit.

    PubMed

    Pette, D; Müller, W; Leisner, E; Vrbová, G

    1976-07-30

    Fast-twitch tibialis anterior and extensor digitorum longus rabbit muscles were subjected to long-term intermittent (8 h daily) or continuous (24 h daily) indirect stimulation with a frequency pattern resembling that of a slow motoneuron. Increases in time to peak of isometric twitch contraction were observed without parallel changes in the pattern of myosin light chains or alterations in the distribution of slow and fast fibres as discernible by the histochemical ATPase reaction. However, changes in the fibre population and in the myosin light chain pattern were observed after intermittent stimulation periods exceeding 40 days or continuous stimulation periods longer than 20 days. Under these conditions even higher increases were found in contraction time. In one animal a complete change in fbire population was observed. In this case myosin light chains of the slow (LCS1, LCS2) and of the fast type (LCf1) were obviously synthetized simultaneously within the same fibre. Early changes in the enzyme activity pattern of energy metabolism indicated a conversion of the fibres including their mitochondrial population. These changes and the earlier reported changes in the sarcoplasmic reticulum are probably responsible for the early changes in contractile properties which occur before the transformation of the myosin. PMID:134352

  15. Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.

    PubMed

    Maruta, S; Homma, K; Ohki, T

    1998-09-01

    Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal muscle myosin containing the highly reactive residues Cys 707 (SH1), Cys 697 (SH2), and lysine 83 (RLR) dramatically alter their local conformation when myosin hydrolyzes ATP, and these changes may reflect formation of a series of transient intermediates during ATP hydrolysis. We used the fluorescent probes 4-fluoro-7-sulfamoylbezofurazan, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and trinitrobenzene-sulfonate, which bind to SH1, SH2, and RLR, respectively, to examine differences in local conformations within myosin.ADP.phosphate analogue (BeFn, Vi, AlF4-, and ScFn) complexes. It was observed that the ternary complexes had SH1 conformations similar to those seen on S-1 in the presence of ATP. In contrast, local conformations in the SH2 and RLR regions of S-1.ADP.BeFn were different from those in corresponding regions of S-1.ADP.AlF4- or ScFn. These results suggest that SH1 and SH2 move distinctly during ATP hydrolysis and that the local conformations of the SH2 and RLR regions more sensitively reflect different transient states. PMID:9722668

  16. Single Molecule Stepping and Structural Dynamics of Myosin X

    PubMed Central

    Sun, Yujie; Sato, Osamu; Ruhnow, Felix; Arsenault, Mark E.; Ikebe, Mitsuo; Goldman, Yale E.

    2010-01-01

    Myosin X is an unconventional myosin with puzzling motility properties. We studied the motility of dimerized myosin X using single molecule fluorescence techniques – polTIRF, FIONA, and Parallax to measure rotation angles and 3-dimensional position of the molecule during its walk. It was found that Myosin X steps processively in a hand-over-hand manner following a left-handed helical path along both single actin filaments and bundles. Its step size and velocity are smaller on actin bundles than individual filaments, suggesting myosin X often steps onto neighboring filaments in a bundle. The data suggest that a previously postulated single α-helical domain mechanically extends the 3-IQ motif lever arm and either the neck-tail hinge or the tail is flexible. These structural features, in conjunction with the membrane and microtubule binding domains, enable myosin X to perform multiple functions on varied actin structures in cells. PMID:20364131

  17. Antiparallel coiled-coil–mediated dimerization of myosin X

    PubMed Central

    Lu, Qing; Ye, Fei; Wei, Zhiyi; Wen, Zilong; Zhang, Mingjie

    2012-01-01

    Processive movements of unconventional myosins on actin filaments generally require motor dimerization. A commonly accepted myosin dimerization mechanism is via formation of a parallel coiled-coil dimer by a stretch of amino acid residues immediately carboxyl-terminal to the motor’s lever-arm domain. Here, we discover that the predicted coiled-coil region of myosin X forms a highly stable, antiparallel coiled-coil dimer (anti-CC). Disruption of the anti-CC either by single-point mutations or by replacement of the anti-CC with a parallel coiled coil with a similar length compromised the filopodial induction activity of myosin X. We further show that the anti-CC and the single α-helical domain of myosin X are connected by a semirigid helical linker. The anti-CC–mediated dimerization may enable myosin X to walk on both single and bundled actin filaments. PMID:23012428

  18. Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.

    PubMed

    Even-Ram, Sharona; Doyle, Andrew D; Conti, Mary Anne; Matsumoto, Kazue; Adelstein, Robert S; Yamada, Kenneth M

    2007-03-01

    Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics. PMID:17310241

  19. Antiparallel coiled-coil-mediated dimerization of myosin X.

    PubMed

    Lu, Qing; Ye, Fei; Wei, Zhiyi; Wen, Zilong; Zhang, Mingjie

    2012-10-23

    Processive movements of unconventional myosins on actin filaments generally require motor dimerization. A commonly accepted myosin dimerization mechanism is via formation of a parallel coiled-coil dimer by a stretch of amino acid residues immediately carboxyl-terminal to the motor's lever-arm domain. Here, we discover that the predicted coiled-coil region of myosin X forms a highly stable, antiparallel coiled-coil dimer (anti-CC). Disruption of the anti-CC either by single-point mutations or by replacement of the anti-CC with a parallel coiled coil with a similar length compromised the filopodial induction activity of myosin X. We further show that the anti-CC and the single α-helical domain of myosin X are connected by a semirigid helical linker. The anti-CC-mediated dimerization may enable myosin X to walk on both single and bundled actin filaments. PMID:23012428

  20. Myosin from striated adductor muscle of Chlamys nipponensis akazara.

    PubMed

    Nishita, K; Ojima, T; Watanabe, S

    1979-09-01

    Myosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant (s 20,2 0 W) and the intrinsic viscosity, [eta] of Akazara myosin thus purified were estimated to be 6.6 S and 2.10 dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of light-chain component (17,000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206,000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1 : 1 : 1. (2) EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin. Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum. PMID:159903

  1. Shared gene structures and clusters of mutually exclusive spliced exons within the metazoan muscle myosin heavy chain genes.

    PubMed

    Kollmar, Martin; Hatje, Klas

    2014-01-01

    Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times within bilaterian evolution. PMID:24498429

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

    PubMed Central

    Kollmar, Martin; Hatje, Klas

    2014-01-01

    Multicellular animals possess two to three different types of muscle tissues. Striated muscles have considerable ultrastructural similarity and contain a core set of proteins including the muscle myosin heavy chain (Mhc) protein. The ATPase activity of this myosin motor protein largely dictates muscle performance at the molecular level. Two different solutions to adjusting myosin properties to different muscle subtypes have been identified so far: Vertebrates and nematodes contain many independent differentially expressed Mhc genes while arthropods have single Mhc genes with clusters of mutually exclusive spliced exons (MXEs). The availability of hundreds of metazoan genomes now allowed us to study whether the ancient bilateria already contained MXEs, how MXE complexity subsequently evolved, and whether additional scenarios to control contractile properties in different muscles could be proposed, By reconstructing the Mhc genes from 116 metazoans we showed that all intron positions within the motor domain coding regions are conserved in all bilateria analysed. The last common ancestor of the bilateria already contained a cluster of MXEs coding for part of the loop-2 actin-binding sequence. Subsequently the protostomes and later the arthropods gained many further clusters while MXEs got completely lost independently in several branches (vertebrates and nematodes) and species (for example the annelid Helobdella robusta and the salmon louse Lepeophtheirus salmonis). Several bilateria have been found to encode multiple Mhc genes that might all or in part contain clusters of MXEs. Notable examples are a cluster of six tandemly arrayed Mhc genes, of which two contain MXEs, in the owl limpet Lottia gigantea and four Mhc genes with three encoding MXEs in the predatory mite Metaseiulus occidentalis. Our analysis showed that similar solutions to provide different myosin isoforms (multiple genes or clusters of MXEs or both) have independently been developed several times within bilaterian evolution. PMID:24498429

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

    PubMed

    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

  4. The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation

    PubMed Central

    Fusi, Luca; Huang, Zhe; Irving, Malcolm

    2015-01-01

    In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads. PMID:26287630

  5. Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments*

    PubMed Central

    Cochran, Jared C.; Thompson, Morgan E.; Kull, F. Jon

    2013-01-01

    G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg2+) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg2+- or Mn2+-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn2+, providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that actoS237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn2+ rescues this effect to near wild-type activity. 2?(3?)-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region. PMID:23960071

  6. Metal switch-controlled myosin II from Dictyostelium discoideum supports closure of nucleotide pocket during ATP binding coupled to detachment from actin filaments.

    PubMed

    Cochran, Jared C; Thompson, Morgan E; Kull, F Jon

    2013-09-27

    G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg(2+)) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg(2+)- or Mn(2+)-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn(2+), providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that actoS237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn(2+) rescues this effect to near wild-type activity. 2'(3')-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region. PMID:23960071

  7. Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

    PubMed

    Zawadowska, B; Majerczak, J; Semik, D; Karasinski, J; Kolodziejski, L; Kilarski, W M; Duda, K; Zoladz, J A

    2004-01-01

    Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training) and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training). Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant differences (P<0.05) regarding composition of muscle fibre types and myosin heavy chains were found only between groups A (41.7+/-1.6% of MyHCI, 40.8+/-4.0% of MyHCIIA and 17.5+/-4.0% of MyHCIIX) and B (64.3+/-0.8% of MyHCI, 34.0+/-1.4% of MyHCIIA and 1.7+/-1.4% of MyHCIIX) and groups A and C (59.6+/-1.6% of MyHCI, 37.2+/-1.3% of MyHCIIA and 3.2+/-1.3% of MyHCIIX). Unexpectedly, endurance athletes (group B) such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance. Furthermore, the relative amount of the fastest MyHCIIX isoform in vastus lateralis muscle was significantly lower in the athletes from group C than in students (group A). We conclude that the myosin profile in the athletes belonging to group C was unfavourable for their sport disciplines. This could be the reason why those athletes did not reach international level despite of several years of training. PMID:15493580

  8. Cargo recognition and cargo-mediated regulation of unconventional myosins.

    PubMed

    Lu, Qing; Li, Jianchao; Zhang, Mingjie

    2014-10-21

    Organized motions are hallmarks of living organisms. Such motions range from collective cell movements during development and muscle contractions at the macroscopic scale all the way down to cellular cargo (e.g., various biomolecules and organelles) transportation and mechanoforce sensing at more microscopic scales. Energy required for these biological motions is almost invariably provided by cellular chemical fuels in the form of nucleotide triphosphate. Biological systems have designed a group of nanoscale engines, known as molecular motors, to convert cellular chemical fuels into mechanical energy. Molecular motors come in various forms including cytoskeleton motors (myosin, kinesin, and dynein), nucleic-acid-based motors, cellular membrane-based rotary motors, and so on. The main focus of this Account is one subfamily of actin filament-based motors called unconventional myosins (other than muscle myosin II, the remaining myosins are collectively referred to as unconventional myosins). In general, myosins can use ATP to fuel two types of mechanomotions: dynamic tethering actin filaments with various cellular compartments or structures and actin filament-based intracellular transport. In contrast to rich knowledge accumulated over many decades on ATP hydrolyzing motor heads and their interactions with actin filaments, how various myosins recognize their specific cargoes and whether and how cargoes can in return regulate functions of motors are less understood. Nonetheless, a series of biochemical and structural investigations in the past few years, including works from our own laboratory, begin to shed lights on these latter questions. Some myosins (e.g., myosin-VI) can function both as cellular transporters and as mechanical tethers. To function as a processive transporter, myosins need to form dimers or multimers. To be a mechanical tether, a monomeric myosin is sufficient. It has been shown for myosin-VI that its cellular cargo proteins can play critical roles in determining the motor properties. Dab2, an adaptor protein linking endocytic vesicles with actin-filament-bound myosin-VI, can induce the motor to form a transport competent dimer. Such a cargo-mediated dimerization mechanism has also been observed in other myosins including myosin-V and myosin-VIIa. The tail domains of myosins are very diverse both in their lengths and protein domain compositions and thus enable motors to engage a broad range of different cellular cargoes. Remarkably, the cargo binding tail of one myosin alone often can bind to multiple distinct target proteins. A series of atomic structures of myosin-V/cargo complexes solved recently reveals that the globular cargo binding tail of the motor contains a number of nonoverlapping target recognition sites for binding to its cargoes including melanophilin, vesicle adaptors RILPL2, and vesicle-bound GTPase Rab11. The structures of the MyTH4-FERM tandems from myosin-VIIa and myosin-X in complex with their respective targets reveal that MyTH4 and FERM domains extensively interact with each other forming structural and functional supramodules in both motors and demonstrate that the structurally similar MyTH4-FERM tandems of the two motors display totally different target binding modes. These structural studies have also shed light on why numerous mutations found in these myosins can cause devastating human diseases such as deafness and blindness, intellectual disabilities, immune disorders, and diabetes. PMID:25230296

  9. Evidence for three fast myosin heavy chain isoforms in type II skeletal muscle fibers in the adult llama (Lama glama).

    PubMed

    Graziotti, G H; Ríos, C M; Rivero, J L

    2001-08-01

    Skeletal muscle fiber types classified on the basis of their content of different myosin heavy chain (MHC) isoforms were analyzed in samples from hindlimb muscles of adult sedentary llamas (Lama glama) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry, and quantitative histochemistry of fiber metabolic and size properties. The immunohistochemical technique allowed the separation of four pure (i.e., expressing a unique MHC isoform) muscle fiber types: one slow-twitch (Type I) and three fast-twitch (Type II) phenotypes. The same four major fiber types could be objectively discriminated with two serial sections stained for mATPase after acid (pH 4.5) and alkaline (pH 10.5) preincubations. The three fast-twitch fiber types were tentatively designated as IIA, IIX, and IIB on the basis of the homologies of their immunoreactivities, acid denaturation of their mATPase activity, size, and metabolic properties expressed at the cellular level with the corresponding isoforms of rat and horse muscles. Acid stability of their mATPase activity increased in the rank order IIA>IIX>IIB. The same was true for size and glycolytic capacity, whereas oxidative capacity decreased in the same rank order IIA>IIX>IIB. In addition to these four pure fibers (I, IIA, IIX, and IIB), four other fiber types with hybrid phenotypes containing two (I+IIA, IIAX, and IIXB) or three (IIAXB) MHCs were immunohistochemically delineated. These frequent phenotypes (40% of the semitendinosus muscle fiber composition) had overlapped mATPase staining intensities with their corresponding pure fiber types, so they could not be delineated by mATPase histochemistry. Expression of the three fast adult MHC isoforms was spatially regulated around islets of Type I fibers, with concentric circles of fibers expressing MHC-IIA, then MHC-IIX, and peripherally MHC-IIB. This study demonstrates that three adult fast Type II MHC isoproteins are expressed in skeletal muscle fibers of the llama. The general assumption that the very fast MHC-IIB isoform is expressed only in small mammals can be rejected. PMID:11457931

  10. Myosin-X: a MyTH-FERM myosin at the tips of filopodia

    PubMed Central

    Kerber, Michael L.; Cheney, Richard E.

    2011-01-01

    Myosin-X (Myo10) is an unconventional myosin with MyTH4-FERM domains that is best known for its striking localization to the tips of filopodia and its ability to induce filopodia. Although the head domain of Myo10 enables it to function as an actin-based motor, its tail contains binding sites for several molecules with central roles in cell biology, including phosphatidylinositol (3,4,5)-trisphosphate, microtubules and integrins. Myo10 also undergoes fascinating long-range movements within filopodia, which appear to represent a newly recognized system of transport. Myo10 is also unusual in that it is a myosin with important roles in the spindle, a microtubule-based structure. Exciting new studies have begun to reveal the structure and single-molecule properties of this intriguing myosin, as well as its mechanisms of regulation and induction of filopodia. At the cellular and organismal level, growing evidence demonstrates that Myo10 has crucial functions in numerous processes ranging from invadopodia formation to cell migration. PMID:22124140

  11. Regulation of myosin II activity by actin architecture

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  12. Myosin filament 3D structure in mammalian cardiac muscle?

    PubMed Central

    AL-Khayat, Hind A.; Morris, Edward P.; Kensler, Robert W.; Squire, John M.

    2008-01-01

    A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2נ430 long, each of which was treated as an independent particle. The resulting 40 resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430 repeat, with successive crown rotations of approximately 60, 60 and 0, rather than the regular 40 for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac). PMID:18472277

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

    PubMed Central

    Brawley, Crista M.; Rock, Ronald S.

    2009-01-01

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

  14. Nonmuscle myosin dependent synthesis of type I collagen

    PubMed Central

    Cai, Le; Fritz, Dillon; Stefanovic, Lela; Stefanovic, Branko

    2010-01-01

    Type I collagen is the most abundant protein in human body synthesized in all tissues as the heterotrimer of two α1(I) and one α2(I) polypeptides. Here we show that intact nonmuscle myosin filaments are required for synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds RNA binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion collagen α1(I) homotrimer, in diminished intracellular colocalization of collagen α1(I) and α2(I) polypeptides, which is required for folding of the heterotrimer, and in their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes and there is a subset of polysomes involved in myosin dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen α1(I) and α2(I) polypeptides. We postulate that LARP6/myosin dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating translation of collagen mRNAs. PMID:20603131

  15. Palytoxin acts on Na+,K+-ATPase but not non-gastric H+,K+-ATPase

    PubMed Central

    Guennoun-Lehmann, Saida; Fonseca, James E.; Horisberger, Jean-Daniel; Rakowski, Robert F.

    2007-01-01

    Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on non-gastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α2- and Na,K-ATPase β2-subunits; Bufo Na,K-ATPase α1- and Na,K-ATPase β2-subunits; and Bufo Na,K-ATPase β2-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μM ouabain. Functional expression was confirmed by measuring 86Rb uptake. PTX (5 nM) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β2-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α1-subunit and Na,K-ATPase β1-subunit; rat Na,K-ATPase α2-subunit and Na,K-ATPase β2-subunit; and rat Na,K-ATPase β1 or Na,K-ATPase β2 subunit alone. Measurement of increases in 86Rb uptake confirmed that both rat Na,K- and H,K-pumps were functional in HeLa cells expressing rat colonic HKα1/NKβ1 and NKα2/NKβ2. Whole-cell patch clamp measurements in HeLa cells expressing rat colonic HKα1/NKβ1 exposed to 100 nM PTX showed no significant increase of membrane current and there was no membrane conductance increase in HeLa cells transfected with rat NKβ1 or rat NKβ2 subunits alone. However, in HeLa Cells expressing rat NKα2 NKβ2, outward current was observed after pump activation by 20 mM K+ and a large membrane conductance increase occurred after 100 nM PTX. We conclude that non-gastric H,K-ATPases are not sensitive to palytoxin when expressed in these cells whereas palytoxin does act on Na,K-ATPase. PMID:17639367

  16. Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

    PubMed Central

    Kampourakis, Thomas; Irving, Malcolm

    2015-01-01

    The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone. PMID:26057075

  17. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

    PubMed

    Duggal, D; Nagwekar, J; Rich, R; Huang, W; Midde, K; Fudala, R; Das, H; Gryczynski, I; Szczesna-Cordary, D; Borejdo, J

    2015-05-15

    Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC. PMID:25770245

  18. 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.

  19. Neuromuscular Development and Regulation of Myosin Expression

    NASA Technical Reports Server (NTRS)

    Bodine, Sue

    1997-01-01

    The proposed experiments were designed to determine whether the absence of gravity during embryogenesis influences the postnatal development of the neuromuscular system. Further, we examined the effects of reduced gravity on hindlimb muscles of the pregnant rats. Microgravity may have short and long-term effects on the development of muscle fiber type differentiation and force producing capabilities. Microgravity will reduce muscle fiber size and cause a shift in myosin heavy chain expression from slow to fast in hindlimb muscles of the adult pregnant rats.

  20. 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.

  1. Tuning myosin-driven sorting on cellular actin networks.

    PubMed

    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. PMID:25738229

  2. Skeletal muscle myosin is the autoantigen for experimental autoimmune myositis.

    PubMed

    Nemoto, Hiroshi; Bhopale, Mahendra K; Constantinescu, Cris S; Schotland, Donald; Rostami, Abdolmohamad

    2003-06-01

    Experimental autoimmune myositis (EAM) is a rodent model for human inflammatory muscle disease (IMD). It can be induced by immunization of rodents with skeletal muscle homogenate and adjuvant. The specific myositogenic autoantigen has not been clearly identified although some evidence points to skeletal muscle myosin. In this report we strengthen this evidence, showing that Lewis rats immunized with purified skeletal muscle myosin develop EAM with the same pattern and severity as EAM induced by whole rabbit skeletal muscle homogenate (WRM). Multiple inflammatory lesions are detected histopathologically in the biceps, quadriceps, and gastrocnemius muscles. Myosin-reactive T cells from animals immunized either with myosin or with WRM have similar patterns of antigen-induced proliferation. The results show that myosin, a component of skeletal muscle, is at least one autoantigen in EAM. PMID:12782010

  3. Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation.

    PubMed

    Ren, Yixin; West-Foyle, Hoku; Surcel, Alexandra; Miller, Christopher; Robinson, Douglas N

    2014-12-15

    How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation. We designed a genetic selection using cDNA library suppression of 3xAsp myosin II to identify factors involved in myosin cleavage furrow accumulation. The 3xAsp mutant is deficient in bipolar thick filament assembly, fails to accumulate at the cleavage furrow, cannot rescue myoII-null cytokinesis, and has impaired mechanosensitive accumulation. Eleven genes suppressed this dominant cytokinesis deficiency when 3xAsp was expressed in wild-type cells. 3xAsp myosin II's localization to the cleavage furrow was rescued by constructs encoding rcdBB, mmsdh, RMD1, actin, one novel protein, and a 14-3-3 hairpin. Further characterization showed that RMD1 is required for myosin II cleavage furrow accumulation, acting in parallel with mechanical stress. Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics. Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp. Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system. PMID:25318674

  4. Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation

    PubMed Central

    Ren, Yixin; West-Foyle, Hoku; Surcel, Alexandra; Miller, Christopher; Robinson, Douglas N.

    2014-01-01

    How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation. We designed a genetic selection using cDNA library suppression of 3xAsp myosin II to identify factors involved in myosin cleavage furrow accumulation. The 3xAsp mutant is deficient in bipolar thick filament assembly, fails to accumulate at the cleavage furrow, cannot rescue myoII-null cytokinesis, and has impaired mechanosensitive accumulation. Eleven genes suppressed this dominant cytokinesis deficiency when 3xAsp was expressed in wild-type cells. 3xAsp myosin II's localization to the cleavage furrow was rescued by constructs encoding rcdBB, mmsdh, RMD1, actin, one novel protein, and a 14-3-3 hairpin. Further characterization showed that RMD1 is required for myosin II cleavage furrow accumulation, acting in parallel with mechanical stress. Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics. Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp. Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system. PMID:25318674

  5. Characterization of vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase in osteoclasts

    SciTech Connect

    Yao, GuanFeng; Feng, HaoTian; Cai, YanLing; Qi, WeiLi; Kong, KangMei . E-mail: kangmeikong@21cn.com

    2007-06-15

    V-ATPase plays important roles in controlling the extra- and intra-cellular pH in eukaryotic cell, which is most crucial for cellular processes. V-ATPases are composed of a peripheral V{sub 1} domain responsible for ATP hydrolysis and integral V{sub 0} domain responsible for proton translocation. Osteoclasts are multinucleated cells responsible for bone resorption and relate to many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone loss. This review summarizes the structure and function of V-ATPase and its subunit, the role of V-ATPase subunits in osteoclast function, V-ATPase inhibitors for osteoclast function, and highlights the importance of V-ATPase as a potential prime target for anti-resorptive agents.

  6. Sodium, potassium-atpases in algae and oomycetes.

    PubMed

    Barrero-Gil, Javier; Garciadeblás, Blanca; Benito, Begoña

    2005-08-01

    We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells. PMID:16167182

  7. Inactivation of mitochondrial ATPase by ultraviolet light

    SciTech Connect

    Chavez, E.; Cuellar, A.

    1984-05-01

    The present work describes experiments that show that far-ultraviolet irradiation induce the inhibition of ATPase activity in both membrane-bound and soluble F1. It was also found that ultraviolet light promotes the release of tightly bound adenine nucleotides from F1-ATPase. Experiments carried out with submitochondrial particles indicate that succinate partially protects against these effects of ultraviolet light. Titration of sulfhydryl groups in both irradiated submitochondrial particles and soluble F1-ATPase indicates that a conformational change induced by photochemical modifications of amino acid residues appears involved in the inactivation of the enzyme. Finally, experiments are described which show that the tyrosine residue located in the active site of F1-ATPase is modified by ultraviolet irradiation.

  8. 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

  9. Phenolic antioxidant scavenging of myosin radicals generated by hypervalent myoglobin.

    PubMed

    Jongberg, Sisse; Lund, Marianne N; Østdal, Henrik; Skibsted, Leif H

    2012-12-01

    The scavenging activity of extracts of green tea (GTE), white grape (WGE), and rosemary (RE), all plant material with high phenolic content, and of the phenolic compounds 4-methylcatechol (4-MC), (+)-catechin, and carnosic acid toward long-lived myosin radicals generated by reaction with H2O2-activated myoglobin at room temperature (pH 7.5, I=1.0) was investigated by freeze-quench ESR spectroscopy. Myosin radicals were generated by incubating 16 μM myosin, 800 μM metmyoglobin, and 800 μM H2O2 for 10 min, and the phenolic extracts were subsequently added (1% (w/w) phenolic compounds relative to myosin). GTE was able to scavenge myosin radicals and reduce the radical intensity by 65%. Furthermore, a low concentration of 4-MC (33 μM) was found to increase the radical concentration when added to the myosin radicals, whereas a higher concentration of 4-MC and catechin (330 μM) was found to scavenge myosin radicals and reduce the overall radical concentration by ∼65%. PMID:23163579

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

    PubMed

    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

    2016-03-29

    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

  11. Stochastic dynamics and mechanosensitivity of myosin II minifilaments

    NASA Astrophysics Data System (ADS)

    Albert, Philipp J.; Erdmann, Thorsten; Schwarz, Ulrich S.

    2014-09-01

    Tissue cells are in a state of permanent mechanical tension that is maintained mainly by myosin II minifilaments, which are bipolar assemblies of tens of myosin II molecular motors contracting actin networks and bundles. Here we introduce a stochastic model for myosin II minifilaments as two small myosin II motor ensembles engaging in a stochastic tug-of-war. Each of the two ensembles is described by the parallel cluster model that allows us to use exact stochastic simulations and at the same time to keep important molecular details of the myosin II cross-bridge cycle. Our simulation and analytical results reveal a strong dependence of myosin II minifilament dynamics on environmental stiffness that is reminiscent of the cellular response to substrate stiffness. For small stiffness, minifilaments form transient crosslinks exerting short spikes of force with negligible mean. For large stiffness, minifilaments form near permanent crosslinks exerting a mean force which hardly depends on environmental elasticity. This functional switch arises because dissociation after the power stroke is suppressed by force (catch bonding) and because ensembles can no longer perform the power stroke at large forces. Symmetric myosin II minifilaments perform a random walk with an effective diffusion constant which decreases with increasing ensemble size, as demonstrated for rigid substrates with an analytical treatment.

  12. The role of myosin phosphorylation in anaphase chromosome movement.

    PubMed

    Sheykhani, Rozhan; Shirodkar, Purnata V; Forer, Arthur

    2013-01-01

    This work deals with the role of myosin phosphorylation in anaphase chromosome movement. Y27632 and ML7 block two different pathways for phosphorylation of the myosin regulatory light chain (MRLC). Both stopped or slowed chromosome movement when added to anaphase crane-fly spermatocytes. To confirm that the effects of the pharmacological agents were on the presumed targets, we studied cells stained with antibodies against mono- or bi-phosphorylated myosin. For all chromosomes whose movements were affected by a drug, the corresponding spindle fibres of the affected chromosomes had reduced levels of 1P- and 2P-myosin. Thus the drugs acted on the presumed target and myosin phosphorylation is involved in anaphase force production. Calyculin A, an inhibitor of MRLC dephosphorylation, reversed and accelerated the altered movements caused by Y27632 and ML-7, suggesting that another phosphorylation pathway is involved in phosphorylation of spindle myosin. Staurosporine, a more general phosphorylation inhibitor, also reduced the levels of MRLC phosphorylation and caused anaphase chromosomes to stop or slow. The effects of staurosporine on chromosome movements were not reversed by Calyculin A, confirming that another phosphorylation pathway is involved in phosphorylation of spindle myosin. PMID:23566798

  13. Structural divergence of the rotary ATPases.

    PubMed

    Muench, Stephen P; Trinick, John; Harrison, Michael A

    2011-08-01

    The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The F₁F(o)-ATPase (F-ATPase) couples ATP synthesis to the electrochemical membrane potential in bacteria, mitochondria and chloroplasts, while the vacuolar H⁺-ATPase (V-ATPase) operates as an ATP-driven proton pump in eukaryotic membranes. In different species of archaea and bacteria, the A₁A(o)-ATPase (A-ATPase) can function as either an ATP synthase or an ion pump. All three of these multi-subunit complexes are rotary molecular motors, sharing a fundamentally similar mechanism in which rotational movement drives the energy conversion process. By analogy to macroscopic systems, individual subunits can be assigned to rotor, axle or stator functions. Recently, three-dimensional reconstructions from electron microscopy and single particle image processing have led to a significant step forward in understanding of the overall architecture of all three forms of these complexes and have allowed the organisation of subunits within the rotor and stator parts of the motors to be more clearly mapped out. This review describes the emerging consensus regarding the organisation of the rotor and stator components of V-, A- and F-ATPases, examining core similarities that point to a common evolutionary origin, and highlighting key differences. In particular, it discusses how newly revealed variation in the complexity of the inter-domain connections may impact on the mechanics and regulation of these molecular machines. PMID:21426606

  14. Myosin motor isoforms direct specification of actomyosin function by tropomyosins

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Murray, George G.; Lord, Matthew

    2015-01-01

    Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in non-muscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this regulation we examined the role of two mammalian tropomyosins (Tpm3.1cy/Tm5NM1 and Tpm4.2cy/Tm4) recently implicated in cancer cell proliferation and metastasis. Like Cdc8p, the Tpm3.1cy and Tpm4.2cy isoforms significantly enhance Myo2p and Myo52p motor activity, converting non-processive Myo52p molecules into processive motors that can walk along actin tracks as single molecules. In contrast to the positive regulation of Myo2p and Myo52p, Cdc8p and the mammalian tropomyosins potently inhibited skeletal muscle myosin-II, while having negligible effects on the highly processive mammalian myosin-Va. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1cy supported normal contractile ring function in fission yeast. Our work reveals that actomyosin regulation by tropomyosin is dependent on the myosin isoform, highlighting a general role for specific isoforms of tropomyosin in sorting myosin motor outputs. PMID:25712463

  15. Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

    PubMed Central

    Hilbert, Lennart; Balassy, Zsombor; Zitouni, Nedjma B.; Mackey, Michael C.; Lauzon, Anne-Marie

    2015-01-01

    Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo—suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state. PMID:25650929

  16. Saturation transfer electron parametric resonance of an indane-dione spin-label. Calibration with hemoglobin and application to myosin rotational dynamics.

    PubMed Central

    Roopnarine, O; Hideg, K; Thomas, D D

    1993-01-01

    We have used a recently synthesized indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione (InVSL) to study the rotational dynamics of myosin, with saturation-transfer electron paramagnetic resonance (ST-EPR). To determine effective rotational correlation times (tau effr) from InVSL spectra, reference spectra corresponding to known correlation times (tau r) were obtained from InVSL-hemoglobin undergoing isotropic rotational motion in aqueous glycerol solutions. These spectra were used to generate plots of spectral parameters vs. tau r. These plots should be used to analyze ST-EPR spectra of InVSL bound to other proteins, because the spectra are different from those of tempo-maleimide-spin-labeled hemoglobin, which have been used previously as ST-EPR standards. InVSL was covalently attached to the head (subfragment-1; S1) of myosin. EPR spectra and K/EDTA-ATPase activity showed that 70-95% of the heads were labeled, with > or = 90% of the label bound to either cys 707 (SH1) or cys 697 (SH2). ST-EPR spectra of InVSL-S1 attached to glass beads, bound to actin in myofibrils, or precipitated with ammonium sulfate indicated no submillisecond rotational motion. Therefore, InVSL is rigidly immobilized on the protein so that it reports the global rotation of the myosin head. The ST-EPR spectra of InVSL-myosin monomers and filaments indicated tau effr values of 4 and 13 microseconds, respectively, showing that myosin heads undergo microsecond segmental rotations that are more restricted in filaments than in monomers. The observed tau effr values are longer than those previously obtained with other spin labels bound to myosin heads, probably because InVSL binds more rigidly to the protein and/or with a different orientation. Further EPR studies of InVSL-myosin in solution and in muscle fibers should prove complementary to previous work with other labels. PMID:8396449

  17. Myosin I Overexpression Impairs Cell Migration

    PubMed Central

    Novak, Kristine D.; Titus, Margaret A.

    1997-01-01

    Dictyostelium myoB, a member of the myosin I family of motor proteins, is important for controlling the formation and retraction of membrane projections by the cell's actin cortex (Novak, K.D., M.D. Peterson, M.C. Reedy, and M.A. Titus. 1995. J. Cell Biol. 131:1205–1221). Mutants that express a three- to sevenfold excess of myoB (myoB+ cells) were generated to further analyze the role of myosin I in these processes. The myoB+ cells move with an instantaneous velocity that is 35% of the wild-type rate and exhibit a 6–8-h delay in initiation of aggregation when placed under starvation conditions. The myoB+ cells complete the developmental cycle after an extended period of time, but they form fewer fruiting bodies that appear to be small and abnormal. The myoB+ cells are also deficient in their ability both to form distinct F-actin filled projections such as crowns and to become elongate and polarized. This defect can be attributed to the presence of at least threefold more myoB at the cortex of the myoB+ cells. In contrast, threefold overexpression of a truncated myoB that lacks the src homology 3 (SH3) domain (myoB/SH3− cells) or myoB in which the consensus heavy chain phosphorylation site was mutated to an alanine (S332A-myoB) does not disturb normal cellular function. However, there is an increased concentration of myoB in the cortex of the myoB/SH3− and S332A-myoB cells comparable to that found in the myoB+ cells. These results suggest that excess full-length cortical myoB prevents the formation of the actin-filled extensions required for locomotion by increasing the tension of the F-actin cytoskeleton and/ or retracting projections before they can fully extend. They also demonstrate a role for the phosphorylation site and SH3 domain in mediating the in vivo activity of myosin I. PMID:9024693

  18. 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 stretch and reach a larger distance. The single-molecule stepping data with mutant full-length construct that lacks two helices out of three in the PT domain tell that it is indeed the case. In this dissertation, more details of myosin VI PT/MT domain experiments will be explored along with background information on the single-molecule experiment methods used in these studies.

  19. Actin cytoskeleton and calcium-ATPase in the process of abomasal mucus secretion in cattle.

    PubMed

    Schessner, M; Schnorr, B

    1990-04-01

    The distribution of actin filaments in pyloric gland cells of cattle was studied with respect to their functional significance in the process of exocrine secretion by use of rhodamine-phalloidin labelling and immunogold-electron microscopy based on the biotin-streptavidin bridge technique. Actin concentrates on the filamentous network of the luminal-cell cortex. Membranes of secretory vesicles accumulating in the cell cortex are also labelled for actin. The present results support the concept of a barrier function of cortical microfilaments entrapping vesicles and linking them to the cytoskeleton. In addition, intracellular localization of calcium-ATPase activity was determined. Enzyme activity associated with the microfilamentous cortical matrix is supposed to be of cytoskeletal nature indicating participation of myosin (-like) structures in the dynamic secretion event. Deposition on the interior aspect of secretory vesicle membranes points to an ATPase transporting calcium into these organelles and enabling them to participate via storage of the cation in intracellular calcium homeostasis, thereby influencing the functional architecture of the cortical cytoskeleton. PMID:2140297

  20. Myofibrillar ATPase activity in skinned human skeletal muscle fibres: fibre type and temperature dependence.

    PubMed Central

    Stienen, G J; Kiers, J L; Bottinelli, R; Reggiani, C

    1996-01-01

    1. Myofibrillar ATP consumption and isometric tension (P0) were determined in chemically skinned skeletal muscle fibres from human rectus abdominis and vastus lateralis muscle. Fibres were classified in four groups (I, IIA, IIB, IIA/B or mixed) based on myosin heavy chain composition. 2. ATP consumption (+/- S.E.M.) at 20 degrees C varied from 0.41 +/- 0.06 mmol l-1 s-1 in type IIB fibres (n = 5) to 0.10 +/- 0.01 mmol l-1 s-1 in type I fibres (n = 13). 3. The ratio between ATPase activity and P0 (tension cost) differed significantly between fast type II and slow type I fibres. At 12 degrees C tension cost was lower than the values found previously in corresponding fibre types in the rat. 4. The relative increase in ATPase activity for a 10 degrees C temperature change (Q10), determined in the range from 12 to 30 degrees C, was temperature independent and amounted to 2.60 +/- 0.06. The increase in P0 with temperature was smaller and declined when the temperature increased. 5. From these measurements, estimates were obtained for the maximum rate of isometric ATP consumption and force development at muscle temperature in vivo (35 degrees C). Images Figure 1 PMID:8782097

  1. Physicochemical changes of myosin and gelling properties of washed tilapia mince as influenced by oxidative stress and microbial transglutaminase.

    PubMed

    Chanarat, Sochaya; Benjakul, Soottawat; Xiong, Youling L

    2015-06-01

    Physicochemical properties of myosin from tilapia subjected to oxidation via Fenton's reaction using H2O2 (0, 0.05, 0.1, 1 and 5 mM) were determined. With increasing H2O2 concentrations and times (from 0 to 12 h), sulfhydryl group content and Ca(2+)-ATPase activity decreased, while carbonyl content and surface hydrophobicity increased to a higher extent. After being subjected to oxidation, cross-linking via disulfide bond along with increased storage modulus (G´) was observed. Microbial transglutaminase (MTGase) induced polymerization of myosin in both non-oxidized and oxidized forms and increased gel G´. Gel properties of washed mince and oxidized washed mince were determined in the presence and absence of MTGase. A stronger gel was observed when 0.3 unit MTGase/g was added, regardless of oxidation process. Nevertheless, the gel strengthening effect of MTGase was hampered when mince was subjected to severe oxidation. Excessive protein aggregation of oxidized samples prior to gelation resulted in the reduction of gel strength and water-holding capacity. Negative effect of protein oxidation on gelation could therefore be alleviated to some degree by MTGase addition. PMID:26028767

  2. Nonmuscle myosin-2: mix and match.

    PubMed

    Heissler, Sarah M; Manstein, Dietmar J

    2013-01-01

    Members of the nonmuscle myosin-2 (NM-2) family of actin-based molecular motors catalyze the conversion of chemical energy into directed movement and force thereby acting as central regulatory components of the eukaryotic cytoskeleton. By cyclically interacting with adenosine triphosphate and F-actin, NM-2 isoforms promote cytoskeletal force generation in established cellular processes like cell migration, shape changes, adhesion dynamics, endo- and exo-cytosis, and cytokinesis. Novel functions of the NM-2 family members in autophagy and viral infection are emerging, making NM-2 isoforms regulators of nearly all cellular processes that require the spatiotemporal organization of cytoskeletal scaffolding. Here, we assess current views about the role of NM-2 isoforms in these activities including the tight regulation of NM-2 assembly and activation through phosphorylation and how NM-2-mediated changes in cytoskeletal dynamics and mechanics affect cell physiological functions in health and disease. PMID:22565821

  3. Principles of Unconventional Myosin Function and Targeting

    PubMed Central

    Hartman, M. Amanda; Finan, Dina; Sivaramakrishnan, Sivaraj; Spudich, James A.

    2016-01-01

    Unconventional myosins are a superfamily of actin-based motors implicated in diverse cellular processes. In recent years, much progress has been made in describing their biophysical properties, and headway has been made into analyzing their cellular functions. Here, we focus on the principles that guide in vivo motor function and targeting to specific cellular locations. Rather than describe each motor comprehensively, we outline the major themes that emerge from research across the superfamily and use specific examples to illustrate each. In presenting the data in this format, we seek to identify open questions in each field as well as to point out commonalities between them. To advance our understanding of myosins’ roles in vivo, clearly we must identify their cellular cargoes and the protein complexes that regulate motor attachment to fully appreciate their functions on the cellular and developmental levels. PMID:21639800

  4. Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

    SciTech Connect

    Minoda, Hiroki; CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 ; Okabe, Tatsuhiro; Inayoshi, Yuhri; Miyakawa, Takuya; Miyauchi, Yumiko; Tanokura, Masaru; Katayama, Eisaku; Wakabayashi, Takeyuki; Akimoto, Tsuyoshi; Sugi, Haruo

    2011-02-25

    Research highlights: {yields} We succeeded in recording structural changes of hydrated myosin cross-bridges. {yields} We succeeded in position-marking the cross-bridges with site-directed antibodies. {yields} We recorded cross-bridge movement at different regions in individual cross-bridge. {yields} The movement was smallest at the cross-bridge-subfragment two boundary. {yields} The results provide evidence for the cross-bridge lever arm mechanism. -- Abstract: Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

  5. Accessibility of Myofilament Cysteines and Effects on ATPase Depend on the Activation State during Exposure to Oxidants

    PubMed Central

    Gross, Sean M.; Lehman, Steven L.

    2013-01-01

    Signaling by reactive oxygen species has emerged as a major physiological process. Due to its high metabolic rate, striated muscle is especially subject to oxidative stress, and there are multiple examples in cardiac and skeletal muscle where oxidative stress modulates contractile function. Here we assessed the potential of cysteine oxidation as a mechanism for modulating contractile function in skeletal and cardiac muscle. Analyzing the cysteine content of the myofilament proteins in striated muscle, we found that cysteine residues are relatively rare, but are very similar between different muscle types and different vertebrate species. To refine this list of cysteines to those that may modulate function, we estimated the accessibility of oxidants to cysteine residues using protein crystal structures, and then sharpened these estimates using fluorescent labeling of cysteines in cardiac and skeletal myofibrils. We demonstrate that cysteine accessibility to oxidants and ATPase rates depend on the contractile state in which preparations are exposed. Oxidant exposure of skeletal and cardiac myofibrils in relaxing solution exposes myosin cysteines not accessible in rigor solution, and these modifications correspond to a decrease in maximum ATPase. Oxidant exposure under rigor conditions produces modifications that increase basal ATPase and calcium sensitivity in ventricular myofibrils, but these effects were muted in fast twitch muscle. These experiments reveal how structural and sequence variations can lead to divergent effects from oxidants in different muscle types. PMID:23894416

  6. Arginylation of myosin heavy chain regulates skeletal muscle strength

    PubMed Central

    Cornachione, Anabelle S.; Leite, Felipe S.; Wang, Junling; Leu, Nicolae A.; Kalganov, Albert; Volgin, Denys; Han, Xuemei; Xu, Tao; Cheng, Yu-Shu; Yates, John R. R.; Rassier, Dilson E.; Kashina, Anna

    2014-01-01

    Protein arginylation is a post-translational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 knockout driven by skeletal muscle-specific creatine kinase (Ckmm) promoter. Such Ckmm-Ate1 mice were viable and outwardly normal, however their skeletal muscle strength was significantly reduced compared to the control. Mass spectrometry of the isolated skeletal myofibrils showed a limited set of proteins arginylated on specific sites, including myosin heavy chain. Atomic force microscopy measurements of the contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of the myosin filaments could be fully rescued by re-arginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in the muscle and exerts a direct effect on muscle strength through arginylation of myosin. PMID:25017061

  7. Emergent Systems Energy Laws for Predicting Myosin Ensemble Processivity

    PubMed Central

    Egan, Paul; Moore, Jeffrey; Schunn, Christian; Cagan, Jonathan; LeDuc, Philip

    2015-01-01

    In complex systems with stochastic components, systems laws often emerge that describe higher level behavior regardless of lower level component configurations. In this paper, emergent laws for describing mechanochemical systems are investigated for processive myosin-actin motility systems. On the basis of prior experimental evidence that longer processive lifetimes are enabled by larger myosin ensembles, it is hypothesized that emergent scaling laws could coincide with myosin-actin contact probability or system energy consumption. Because processivity is difficult to predict analytically and measure experimentally, agent-based computational techniques are developed to simulate processive myosin ensembles and produce novel processive lifetime measurements. It is demonstrated that only systems energy relationships hold regardless of isoform configurations or ensemble size, and a unified expression for predicting processive lifetime is revealed. The finding of such laws provides insight for how patterns emerge in stochastic mechanochemical systems, while also informing understanding and engineering of complex biological systems. PMID:25885169

  8. Walking mechanism of the intracellular cargo transporter myosin V

    NASA Astrophysics Data System (ADS)

    Schmitz, Stephan; Smith-Palmer, Jayne; Sakamoto, Takeshi; Sellers, James R.; Veigel, Claudia

    2006-08-01

    Motor proteins of the myosin, kinesin and dynein families transport vesicles and other cargo along tracks of actin filaments or microtubules through the cytoplasm of cells. The mechanism by which myosin V, a motor involved in several types of intracellular transport, moves processively along actin filaments, has recently been the subject of many single molecule biophysical studies. Details of the molecular mechanisms by which this molecular motor operates are starting to emerge.

  9. Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.

    1996-01-01

    Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

  10. Contribution of myosin II activity to cell spreading dynamics.

    PubMed

    Nisenholz, Noam; Paknikar, Aishwarya; Köster, Sarah; Zemel, Assaf

    2016-01-14

    Myosin II activity and actin polymerization at the leading edge of the cell are known to be essential sources of cellular stress. However, a quantitative account of their separate contributions is still lacking; so is the influence of the coupling between the two phenomena on cell spreading dynamics. We present a simple analytic elastic theory of cell spreading dynamics that quantitatively demonstrates how actin polymerization and myosin activity cooperate in the generation of cellular stress during spreading. Consistent with experiments, myosin activity is assumed to polarize in response to the stresses generated during spreading. The characteristic response time and the overall spreading time are predicted to determine different evolution profiles of cell spreading dynamics. These include, a (regular) monotonic increase of cell projected area with time, a non-monotonic (overshooting) profile with a maximum, and damped oscillatory modes. In addition, two populations of myosin II motors are distinguished based on their location in the lamella; those located above the major adhesion zone at the cell periphery are shown to facilitate spreading whereas those in deeper regions of the lamella are shown to oppose spreading. We demonstrate that the attenuation of myosin activity in the two regions may result in reciprocal effects on spreading. These findings provide important new insight into the function of myosin II motors in the course of spreading. PMID:26481613

  11. An invertebrate smooth muscle with striated muscle myosin filaments

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-20

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

  13. Control of myosin-I force sensing by alternative splicing.

    PubMed

    Laakso, Joseph M; Lewis, John H; Shuman, Henry; Ostap, E Michael

    2010-01-12

    Myosin-Is are molecular motors that link cellular membranes to the actin cytoskeleton, where they play roles in mechano-signal transduction and membrane trafficking. Some myosin-Is are proposed to act as force sensors, dynamically modulating their motile properties in response to changes in tension. In this study, we examined force sensing by the widely expressed myosin-I isoform, myo1b, which is alternatively spliced in its light chain binding domain (LCBD), yielding proteins with lever arms of different lengths. We found the actin-detachment kinetics of the splice isoforms to be extraordinarily tension-sensitive, with the magnitude of tension sensitivity to be related to LCBD splicing. Thus, in addition to regulating step-size, motility rates, and myosin activation, the LCBD is a key regulator of force sensing. We also found that myo1b is substantially more tension-sensitive than other myosins with similar length lever arms, indicating that different myosins have different tension-sensitive transitions. PMID:20080738

  14. The dynamic stator stalk of rotary ATPases

    PubMed Central

    Stewart, Alastair G.; Lee, Lawrence K.; Donohoe, Mhairi; Chaston, Jessica J.; Stock, Daniela

    2012-01-01

    Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases. PMID:22353718

  15. Conformational flexibility of loops of myosin enhances the global bias in the actin-myosin interaction landscape.

    PubMed

    Nie, Qing-Miao; Sasai, Masaki; Terada, Tomoki P

    2014-04-14

    A long-standing controversy on the mechanism of an actomyosin motor is the role of the Brownian motion of the myosin head in force generation. In order to shed light on this problem, we calculate free-energy landscapes of interaction between an actin filament and the head (S1) of myosin II by using a coarse-grained model of actomyosin. The results show that the free-energy landscape has a global gradient toward the strong-binding site on actin filament, which explains the biased Brownian motion of myosin S1 observed in a single-molecule experiment [Kitamura et al., Nature, 1999, 397, 129 and Biophysics, 2005, 1, 1]. The distinct global gradient in the landscape is brought about only when the conformation of loop 2 at the actin interface of myosin S1 is flexible. The conformational flexibility of loop 3 also contributes to the gradient in the landscape by compensating the role of loop 2. Though the structure of loop 2 is expanded in the weak-binding state, loop 2 shows the larger fluctuation of compaction and expansion due to the actin-myosin interactions as myosin S1 moves toward the strong-binding site on actin filament. Hence, the increase in the compaction-expansion fluctuation of loop 2, the stronger binding of myosin to actin, and the biased Brownian motion of myosin S1 are coupled with each other and should take place in a concurrent way. This predicted coupling should provide opportunities to further test the hypothesis of the biased Brownian motion in actomyosin. PMID:24513657

  16. Changes in myosin isozyme expression during cardiac hypertrophy in hyperthyroid rabbits.

    PubMed

    Seiden, D; Srivatsan, M; Navidad, P A

    1989-01-01

    The myosin isozyme distribution in the left ventricle and in the interventricular septum of rabbits was studied after 3, 7, 11, 14 and 21 days of L-thyroxine (500 micrograms/kg/day) administration. Histochemical procedures were employed to identify V1 and V3 by their Ca2+ ATPase activity and their proportions were quantified through polyacrylamide gel electrophoresis. In the left ventricle, the subepicardium was the first to show the shift from V3 to V1, followed by the subendocardium. The intermediate region became heterogeneous by 11 days and remained so until 21 days. The right subendocardial and the intermediate regions of the interventricular septum were heterogeneous in the normal rabbit and hyperthyroidism resulted in a shift from V3 to V1 in both the right and left subendocardial regions of the septum. Like the left ventricle, the intermediate region of the interventricular septum remained heterogeneous. Localized accumulations of collagen were seen in all regions of the left ventricle and interventricular septum. From these results we conclude that in thyrotoxic myocardial hypertrophy the isozymic shift from V3 to V1 is progressive, region-specific and is directly correlated with the period of hyperthyroidism in the first 2 weeks. Prolonged hyperthyroidism results in localized accumulation of collagen which does not exhibit any regional specificity. PMID:2528881

  17. Biosynthesis of the tonoplast H sup + -ATPase

    SciTech Connect

    Randall, S.K. ); Sze, H. )

    1989-04-01

    To determine whether the tonoplast H{sup +}-ATPase was differentially synthesized in oat seedlings, sections were labeled in vivo with ({sup 35}S)-methionine and ATPase subunits were immunoprecipitated. Subunits were detected in all portions of the seedling with the exception of the seed. The intracellular site of synthesis for two peripheral ATPase subunits was investigated. RNA encoding the 72 kDa (catalytic) subunit was found in membrane-bound polysomes. In contrast, message for the 60 kDa subunit was found on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences, suggesting the absence of cleaved precursors for the 72 or 60 kDa subunits.

  18. ATPase Activity Measurements Using Radiolabeled ATP.

    PubMed

    Swarts, Herman G P; Koenderink, Jan B

    2016-01-01

    ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [γ-(32)P]ATP, which is hydrolyzed by P-type ATPases to ADP and (32)Pi. Activated charcoal is used to bind the excess of [γ-(32)P]ATP, which can be separated from the unbound (32)Pi by centrifugation. With this method, a wide range (0.1 μM-10 mM) of ATP can be used. In addition, we also describe in detail how ATP hydrolysis is translated into ATPase activity. PMID:26695028

  19. 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.

  20. ATPases, ion exchangers and human sperm motility.

    PubMed

    Peralta-Arias, Rubén D; Vívenes, Carmen Y; Camejo, María I; Piñero, Sandy; Proverbio, Teresa; Martínez, Elizabeth; Marín, Reinaldo; Proverbio, Fulgencio

    2015-05-01

    Human sperm has several mechanisms to control its ionic milieu, such as the Na,K-ATPase (NKA), the Ca-ATPase of the plasma membrane (PMCA), the Na(+)/Ca(2) (+)-exchanger (NCX) and the Na(+)/H(+)-exchanger (NHE). On the other hand, the dynein-ATPase is the intracellular motor for sperm motility. In this work, we evaluated NKA, PMCA, NHE, NCX and dynein-ATPase activities in human sperm and investigated their correlation with sperm motility. Sperm motility was measured by Computer Assisted Semen Analysis. It was found that the NKA activity is inhibited by ouabain with two Ki (7.9 × 10(-9) and 9.8 × 10(-5) M), which is consistent with the presence of two isoforms of α subunit of the NKA in the sperm plasma membranes (α1 and α4), being α4 more sensitive to ouabain. The decrease in NKA activity is associated with a reduction in sperm motility. In addition, sperm motility was evaluated in the presence of known inhibitors of NHE, PMCA and NCX, such as amiloride, eosin, and KB-R7943, respectively, as well as in the presence of nigericin after incubation with ouabain. Amiloride, eosin and KB-R7943 significantly reduced sperm motility. Nigericin reversed the effect of ouabain and amiloride on sperm motility. Dynein-ATPase activity was inhibited by acidic pH and micromolar concentrations of Ca(2) (+). We explain our results in terms of inhibition of the dynein-ATPase in the presence of higher cytosolic H(+) and Ca(2) (+), and therefore inhibition of sperm motility. PMID:25820902

  1. [Molecular and functional diversity of NA,K-ATPase and renal H,K-ATPases].

    PubMed

    Jaisser, F

    1996-01-01

    Potassium homeostasis is a determinant factor in the maintenance of many vital functions. Cell excitability, for instance, in striate and cardiac muscle, as well as in neurons, is dependent upon the ratio of potassium levels on either side of the plasmic membrane. Acute or chronic mechanisms of adjustment to disorders of bodily potassium balance exist in muscle, the kidney and distal colon. Na+K(+)-ATPase is involved in potassium transfers between the extracellular and intracellular compartments, in particular in muscle, enabling the creation of an appropriate trans-membrane K gradient. Na+K(+)-ATPase also participates in the development and maintenance of a transmembrane potassium electrochemical gradient necessary for potassium secretion processes in the kidney or distal colon. Colonic and renal H+K(+)-ATPases, so-called non-gastric H+K(+)-ATPases, are involved in the absorption of potassium from the gastrointestinal lumen or urinary fluid. They have an important role to play during chronic disorders, e.g. chronic bodily potassium depletion. Renal H+K(+)-ATPases and Na+K-ATPase are P-ATPases, consisting of a heterodimer of two alpha and beta sub-units. Several isoforms have been identified, on both a molecular and functional basis, for both the alpha and beta sub-unit. These two ATPases form part of the Na+K(+)-ATPase/H+K(+)-ATPase gene group. These pumps share many structural and functional similarities, but also particular functional specificities, probably involved in separate physiological roles for each isoform. Four isoforms of the alpha sub-unit and two isoforms of the beta sub-unit of Na+K(+)-ATPase have been identified. Sensitivity to ouabain, a Na+K(+)-ATPase inhibitor, differs according to the alpha isoform present in the alpha beta heterodimer. It is also involved in the catalytic cycle and influences pump potassium affinity. Several H+K(+)-ATPases have been identified from a molecular standpoint: gastric H+K(+)-ATPases and a colonic H+K(+)-ATPase found more recently. Recent studies have shown that both these H+K(+)-ATPases exist in the kidney. "Gastric" H+K(+)-ATPase is active along the entire length of the collecting tubule, in rats exposed to a normal potassium intake. In contrast, colonic H+K(+)-ATPase is active only in the cells of the external medullary collecting duct. This activity cannot be detected in animals on a standard diet but is very powerfully induced by potassium depletion. Activity is independent of steroidal status and of aldosterone in particular. Identification of a molecular homologue in the bladder of the amphibian Bufo marinus (the functional equivalent of the cortical collecting duct of mammals) has enabled the development of functional tests by activity in the oocyte of Xenopus laevis. The use this functional approach has shown that bladder H+K(+)-ATPase, just like that of rat distal colon, is sensitive to ouabain, an inhibitor considered up to now to be specific to Na+K(+)-ATPase. In contrast, this H+K(+)-ATPase shows little or no sensitivity to Sch 28080, a "classical" gastric H+K(+)-ATPase inhibitor. It thus seems that two H+K(+)-ATPases, different from a molecular standpoint, exist in rat kidney. They differ in terms of their cellular activity, regulation and functional properties. This is strongly suggestive of a specific role of each of them in potassium homeostasis, a role which remains to be defined. The use of genetically modified animals, as well as of physiological studies more focussed on this question, should provide clarification of the specific functional role of each isoform of the alpha and beta sub-units of renal H+K(+)-ATPases and Na+K(+)-ATPase. Extrapolation of these results to human pathophysiology is quite another challenge. Control of Na+K(+)-ATPase activity by endoouabain and its effects on cardiovascular pathophysiology must be identified. An H+K(+)-ATPase with molecular and functional characteristics similar to those of amphibian bladder and rat colon H+K(+)-A PMID:9019667

  2. Myocardial Na,K-ATPase: Clinical aspects

    PubMed Central

    Kjeldsen, Keld

    2003-01-01

    The specific binding of digitalis glycosides to Na,K-ATPase is used as a tool for Na,K-ATPase quantification with high accuracy and precision. In myocardial biopsies from patients with heart failure, total Na,K-ATPase concentration is decreased by around 40%; a correlation exists between a decrease in heart function and a decrease in Na,K-ATPase concentration. During digitalization, around 30% of remaining pumps are occupied by digoxin. Myocardial Na,K-ATPase is also influenced by other drugs used for the treatment of heart failure. Thus, potassium loss during diuretic therapy has been found to reduce myocardial Na,K-ATPase, whereas angiotensin-converting enzyme inhibitors may stimulate Na,K pump activity. Furthermore, hyperaldosteronism induced by heart failure has been found to decrease Na,K-ATPase activity. Accordingly, treatment with the aldosterone antagonist, spironolactone, may also influence Na,K-ATPase activity. The importance of Na,K pump modulation with heart disease, inhibition in digitalization and other effects of medication should be considered in the context of sodium, potassium and calcium regulation. It is recommended that digoxin be administered to heart failure patients who, after institution of mortality-reducing therapy, still have heart failure symptoms, and that the therapy be continued if symptoms are revealed or reduced. Digitalis glycosides are the only safe inotropic drugs for oral use that improve hemodynamics in heart failure. An important aspect of myocardial Na,K pump affection in heart disease is its influence on extracellular potassium (Ke) homeostasis. Two important aspects should be considered: potassium handling among myocytes, and effects of potassium entering the extracellular space of the heart via the bloodstream. It should be noted that both of these aspects of Ke homeostasis are affected by regulatory aspects, eg, regulation of the Na,K pump by physiological and pathophysiological conditions, as well as by medical treatments. Digitalization has been shown to affect both parameters. Furthermore, in experimental animals, potassium loading and depletion are found to significantly affect Ke handling. The effects of potassium depletion are of special interest because this condition often occurs in patients treated with diuretics. In human congenital long QT syndrome caused by mutations in genes coding for potassium channels, exercise and potassium depletion are well known for their potential to elicit arrhythmias and sudden death. There is a need for further evaluation of the dynamic aspects of potassium handling in the heart, as well as in the periphery. It is recommended that resting plasma potassium be maintained at around 4 mmol/L. PMID:19641704

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

    PubMed

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

    1989-03-01

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

  4. Comprehensive physical mechanism of two-headed biomotor myosin V

    NASA Astrophysics Data System (ADS)

    Xu, Yuzhi; Wang, Zhisong

    2009-12-01

    Two-headed biomotor myosin V autonomously coordinates its two identical heads in fuel consumption and mechanical stepping, so that the dimerized motor as a whole gains the capability of processive, unidirectional movement along cytoskeletal filament. How the dimer-level functions like sustained direction rectification and autonomous coordination emerge out of physical principles poses an outstanding question pertinent to motor protein biology as well as the nascent field of bioinspired nanomotors. Here the comprehensive physical mechanism for myosin V motor is identified by a dimer-level free-energy analysis that is methodologically calibrated against experimental data. A hallmark of the identified mechanism is a mechanically mediated symmetry breaking that occurs at the dimer level and prevails against ubiquitous thermal fluctuations. Another character is the onset of substantial free-energy gaps between major dimer-track binding configurations. The symmetry breaking is the basis for myosin V's directional rectification, and the energy gaps facilitate autonomous head-head coordination. The mechanism explains the experimental finding that myosin V makes ATP-independent consecutive steps under high opposing loads but not under pushing loads. Interestingly, myosin V and another major biomotor kinesin 1 are found to share essentially the same core mechanism but for distinctly different working regimes.

  5. Collective dynamics of elastically coupled myosin V motors.

    PubMed

    Lu, Hailong; Efremov, Artem K; Bookwalter, Carol S; Krementsova, Elena B; Driver, Jonathan W; Trybus, Kathleen M; Diehl, Michael R

    2012-08-10

    Characterization of the collective behaviors of different classes of processive motor proteins has become increasingly important to understand various intracellular trafficking and transport processes. This work examines the dynamics of structurally-defined motor complexes containing two myosin Va (myoVa) motors that are linked together via a molecular scaffold formed from a single duplex of DNA. Dynamic changes in the filament-bound configuration of these complexes due to motor binding, stepping, and detachment were monitored by tracking the positions of different color quantum dots that report the position of one head of each myoVa motor on actin. As in studies of multiple kinesins, the run lengths produced by two myosins are only slightly larger than those of single motor molecules. This suggests that internal strain within the complexes, due to asynchronous motor stepping and the resultant stretching of motor linkages, yields net negative cooperative behaviors. In contrast to multiple kinesins, multiple myosin complexes move with appreciably lower velocities than a single-myosin molecule. Although similar trends are predicted by a discrete state stochastic model of collective motor dynamics, these analyses also suggest that multiple myosin velocities and run lengths depend on both the compliance and the effective size of their cargo. Moreover, it is proposed that this unique collective behavior occurs because the large step size and relatively small stalling force of myoVa leads to a high sensitivity of motor stepping rates to strain. PMID:22718762

  6. Dicyclohexylcarbodiimide-sensitive ATPase in Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Kristjansson, H.; Hochstein, L. I.

    1985-01-01

    Membranes from Halobacterium saccharovorum contained a cryptic ATPase which required Mg(2+) or Mn(2+) and was activated by Triton X-100. The optimal pH for ATP hydrolysis was 9-10. ATP or GTP were hydrolyzed at the same rate while ITP, CTP, and UTP were hydrolyzed at about half that rate. The products of ATP hydrolysis were ADP and phosphate. The ATPase required high concentrations (3.5 M) of NaCl for maximum activity. ADP was a competitive inhibitor of the activity, with an apparent Ki of 50 micro-M. Dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis. The inhibition was marginal at the optimum pH of the enzyme. When the ATPase was preincubated with DCCD at varying pH values, but assayed at the optimal pH for activity, DCCD inhibition was observed to increase with increasing acidity of the preincubation medium. DCCD inhibition was also dependent on time of preincubation, and protein and DCCD concentrations. When preincubated at pH 6.0 for 4 h at a protein:DCCD ratio of 40 (w/w), ATPase activity was inhibited 90 percent.

  7. Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

    PubMed

    Newman-Smith, Erin; Kourakis, Matthew J; Reeves, Wendy; Veeman, Michael; Smith, William C

    2015-01-01

    The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization. PMID:25866928

  8. Functions of plant-specific myosin XI: from intracellular motility to plant postures.

    PubMed

    Ueda, Haruko; Tamura, Kentaro; Hara-Nishimura, Ikuko

    2015-12-01

    The plant-specific protein motor class myosin XI is known to function in rapid bulk flow of the cytoplasm (cytoplasmic streaming) and in organellar movements. Recent studies unveiled a wide range of physiological functions of myosin XI motors, from intracellular motility to organ movements. Arabidopsis thaliana has 13 members of myosin XI class. In vegetative organs, myosins XIk, XI1, and XI2 primarily contribute to dynamics and spatial configurations of endoplasmic reticulum that develops a tubular network in the cell periphery and thick strand-like structures in the inner cell regions. Myosin XI-i forms a nucleocytoplasmic linker and is responsible for nuclear movement and shape. In addition to these intracellular functions, myosin XIf together with myosin XIk is involved in the fundamental nature of plants; the actin-myosin XI cytoskeleton regulates organ straightening to adjust plant posture. PMID:26432645

  9. Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle.

    PubMed

    Wu, X; Rao, K; Bowers, M B; Copeland, N G; Jenkins, N A; Hammer, J A

    2001-03-01

    The peripheral accumulation of melanosomes characteristic of wild-type mouse melanocytes is driven by a cooperative process involving long-range, bidirectional, microtubule-dependent movements coupled to capture and local movement in the actin-rich periphery by myosin Va, the product of the dilute locus. Genetic evidence suggests that Rab27a, the product of the ashen locus, functions with myosin Va in this process. Here we show that ashen melanocytes, like dilute melanocytes, exhibit normal dendritic morphology and melanosome biogenesis, an abnormal accumulation of end-stage melanosomes in the cell center, and rapid, bidirectional, microtubule-dependent melanosome movements between the cell center and the periphery. This phenotype suggests that ashen melanocytes, like dilute melanocytes, are defective in peripheral melanosome capture. Consistent with this, introduction into ashen melanocytes of cDNAs encoding wild-type and GTP-bound versions of Rab27a restores the peripheral accumulation of melanosomes in a microtubule-dependent manner. Conversely, introduction into wild-type melanocytes of the GDP-bound version of Rab27a generates an ashen/dilute phenotype. Rab27a colocalizes with end-stage melanosomes in wild-type cells, and is most concentrated in melanosome-rich dendritic tips, where it also colocalizes with myosin Va. Finally, neither endogenous myosin Va nor an expressed, GFP-tagged, myosin Va tail domain fusion protein colocalize with melanosomes in ashen melanocytes, in contrast to that seen previously in wild-type cells. These results argue that Rab27a serves to enable the myosinVa-dependent capture of melanosomes delivered to the periphery by bidirectional, microtubule-dependent transport, and that it does so by recruiting the myosin to the melanosome surface. We suggest that Rab27a, in its GTP-bound and melanosome-associated form, predominates in the periphery, and that it is this form that recruits the myosin, enabling capture. These results argue that Rab27a serves as a myosin Va 'receptor', and add to the growing evidence that Rab GTPases regulate vesicle motors as well as SNARE pairing. PMID:11228153

  10. 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. PMID:26607798

  11. Coarse-Grained Simulation of Myosin-V Movement

    PubMed Central

    Katsimitsoulia, Zoe; Taylor, William R.

    2012-01-01

    We describe the development of a hierarchic modelling method applied to simulating the processive movement of the myosin-V molecular motor protein along an actin filament track. In the hierarchic model, three different levels of protein structure resolution are represented: secondary structure, domain, and protein, with the level of detail changing according to the degree of interaction among the molecules. The integrity of the system is maintained using a tree of spatially organised bounding volumes and distance constraints. Although applied to an actin-myosin system, the hierarchic framework is general enough so that it may easily be adapted to a number of other large biomolecular systems containing in the order of 100 proteins. We compared the simulation results with biophysical data, and despite the lack of atomic detail in our model, we find good agreement and can even suggest some refinements to the current model of myosin-V motion. PMID:22675402

  12. Expression, Splicing, and Evolution of the Myosin Gene Family in Plants1[W][OA

    PubMed Central

    Peremyslov, Valera V.; Mockler, Todd C.; Filichkin, Sergei A.; Fox, Samuel E.; Jaiswal, Pankaj; Makarova, Kira S.; Koonin, Eugene V.; Dolja, Valerian V.

    2011-01-01

    Plants possess two myosin classes, VIII and XI. The myosins XI are implicated in organelle transport, filamentous actin organization, and cell and plant growth. Due to the large size of myosin gene families, knowledge of these molecular motors remains patchy. Using deep transcriptome sequencing and bioinformatics, we systematically investigated myosin genes in two model plants, Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon). We improved myosin gene models and found that myosin genes undergo alternative splicing. We experimentally validated the gene models for Arabidopsis myosin XI-K, which plays the principal role in cell interior dynamics, as well as for its Brachypodium ortholog. We showed that the Arabidopsis gene dubbed HDK (for headless derivative of myosin XI-K), which emerged through a partial duplication of the XI-K gene, is developmentally regulated. A gene with similar architecture was also found in Brachypodium. Our analyses revealed two predominant patterns of myosin gene expression, namely pollen/stamen-specific and ubiquitous expression throughout the plant. We also found that several myosins XI can be rhythmically expressed. Phylogenetic reconstructions indicate that the last common ancestor of the angiosperms possessed two myosins VIII and five myosins XI, many of which underwent additional lineage-specific duplications. PMID:21233331

  13. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

    PubMed

    Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

    2016-04-01

    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

  14. A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle.

    PubMed

    Gambke, B; Rubinstein, N A

    1984-10-10

    A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development. PMID:6384219

  15. Chicken myosin IB mRNA is highly expressed in lymphoid tissues.

    PubMed Central

    Edgar, A J; Knight, A E; Bennett, J P

    1996-01-01

    Little is known about the functions of members of the myosin I family in vertebrates. Chicken myosin IB is a member of the amoeba-type subclass of myosin I molecules and tissue localisation studies may provide possible clues to the functions of these myosin I molecules. The expression of the mRNA of this unconventional myosin IB was analysed by in situ hybridization and compared with that of the well characterised brush border myosin I on frozen sections of tissues from the adult domestic chicken. High levels of myosin IB mRNA were found in the intestine and spleen, but were not found in other tissues examined such as brain, heart, lung, liver and kidney. In the intestine, myosin IB mRNA was much more abundant in the lamina propria than in the enterocytes, whereas brush border myosin I mRNA was restricted to the enterocytes. In the spleen, myosin IB mRNA expression was abundant in regions of white pulp, namely germinal centres, periellipsoid lymphocyte sheaths and periarteriolar lymphocyte sheaths. Lymphocytes are the major cell type in both the lamina propria and the white pulp of the spleen, which suggests that chicken myosin IB is highly expressed in lymphocytes. Lymphocyte recirculation depends on their migration through the endothelial layer and it is possible that myosin IB may have a role to play in this type of cell motility. Images Fig. 1 Fig. 2 Fig. 3 PMID:8886967

  16. 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

  17. Effect of N-Terminal Extension of Cardiac Troponin I on the Ca(2+) Regulation of ATP Binding and ADP Dissociation of Myosin II in Native Cardiac Myofibrils.

    PubMed

    Gunther, Laura K; Feng, Han-Zhong; Wei, Hongguang; Raupp, Justin; Jin, Jian-Ping; Sakamoto, Takeshi

    2016-03-29

    Cardiac troponin I (cTnI) has a unique N-terminal extension that plays a role in modifying the calcium regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions as a physiological adaptation. Recent studies have shown that in comparison with controls, transgenic mouse cardiac myofibrils containing cTnI lacking the N-terminal extension (cTnI-ND) had a lower sensitivity to calcium activation of ATPase, resulting in enhanced ventricular relaxation and cardiac function. To investigate which step(s) of the ATPase cycle is regulated by the N-terminal extension of cTnI, here we studied the calcium dependence of cardiac myosin II ATPase kinetics in isolated cardiac myofibrils. ATP binding and ADP dissociation rates were measured by using stopped-flow spectrofluorimetry with mant-dATP and mant-dADP, respectively. We found that the second-order mant-dATP binding rate of cTnI-ND mouse cardiac myofibrils was 3-fold faster than that of wild-type myofibrils at low Ca(2+) concentrations. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentration, while the wild-type controls were not significantly affected. These data from experiments using native cardiac myofibrils under physiological conditions indicate that modification of the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase. PMID:26862665

  18. Myopathy-causing Q147P TPM2 mutation shifts tropomyosin strands further towards the open position and increases the proportion of strong-binding cross-bridges during the ATPase cycle.

    PubMed

    Karpicheva, Olga E; Simonyan, Armen O; Kuleva, Nadezhda V; Redwood, Charles S; Borovikov, Yurii S

    2016-03-01

    The molecular mechanisms of skeletal muscle dysfunction in congenital myopathies remain unclear. The present study examines the effect of a myopathy-causing mutation Q147P in β-tropomyosin on the position of tropomyosin on troponin-free filaments and on the actin–myosin interaction at different stages of the ATP hydrolysis cycle using the technique of polarized fluorimetry. Wild-type and Q147P recombinant tropomyosins, actin, and myosin subfragment-1 were modified by 5-IAF, 1,5-IAEDANS or FITC-phalloidin, and 1,5-IAEDANS, respectively, and incorporated into single ghost muscle fibers, containing predominantly actin filaments which were free of troponin and tropomyosin. Despite its reduced affinity for actin in co-sedimentation assay, the Q147P mutant incorporates into the muscle fiber. However, compared to wild-type tropomyosin, it locates closer to the center of the actin filament. The mutant tropomyosin increases the proportion of the strong-binding myosin heads and disrupts the co-operation of actin and myosin heads during the ATPase cycle. These changes are likely to underlie the contractile abnormalities caused by this mutation. PMID:26708479

  19. Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes.

    PubMed

    Baker, Nicola; Hamilton, Graham; Wilkes, Jonathan M; Hutchinson, Sebastian; Barrett, Michael P; Horn, David

    2015-07-21

    Kinetoplastid parasites cause lethal diseases in humans and animals. The kinetoplast itself contains the mitochondrial genome, comprising a huge, complex DNA network that is also an important drug target. Isometamidium, for example, is a key veterinary drug that accumulates in the kinetoplast in African trypanosomes. Kinetoplast independence and isometamidium resistance are observed where certain mutations in the F1-γ-subunit of the two-sector F1Fo-ATP synthase allow for Fo-independent generation of a mitochondrial membrane potential. To further explore kinetoplast biology and drug resistance, we screened a genome-scale RNA interference library in African trypanosomes for isometamidium resistance mechanisms. Our screen identified 14 V-ATPase subunits and all 4 adaptin-3 subunits, implicating acidic compartment defects in resistance; V-ATPase acidifies lysosomes and related organelles, whereas adaptin-3 is responsible for trafficking among these organelles. Independent strains with depleted V-ATPase or adaptin-3 subunits were isometamidium resistant, and chemical inhibition of the V-ATPase phenocopied this effect. While drug accumulation in the kinetoplast continued after V-ATPase subunit depletion, acriflavine-induced kinetoplast loss was specifically tolerated in these cells and in cells depleted for adaptin-3 or endoplasmic reticulum membrane complex subunits, also identified in our screen. Consistent with kinetoplast dispensability, V-ATPase defective cells were oligomycin resistant, suggesting ATP synthase uncoupling and bypass of the normal Fo-A6-subunit requirement; this subunit is the only kinetoplast-encoded product ultimately required for viability in bloodstream-form trypanosomes. Thus, we describe 30 genes and 3 protein complexes associated with kinetoplast-dependent growth. Mutations affecting these genes could explain natural cases of dyskinetoplasty and multidrug resistance. Our results also reveal potentially conserved communication between the compartmentalized two-sector rotary ATPases. PMID:26150481

  20. The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states.

    PubMed

    Ray, Tushar

    2013-01-01

    This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump. PMID:24555080

  1. The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states

    PubMed Central

    Ray, Tushar

    2013-01-01

    This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump. PMID:24555080

  2. P2C-Type ATPases and Their Regulation.

    PubMed

    Retamales-Ortega, Rocío; Vio, Carlos P; Inestrosa, Nibaldo C

    2016-03-01

    P2C-type ATPases are a subfamily of P-type ATPases comprising Na(+)/K(+)-ATPase and H(+)/K(+)-ATPase. Na(+)/K(+)-ATPase is ubiquitously expressed and has been implicated in several neurological diseases, whereas H(+)/K(+)-ATPase is found principally in the colon, stomach, and kidney. Both ATPases have two subunits, α and β, but Na(+)/K(+)-ATPase also has a regulatory subunit called FXYD, which has an important role in cancer. The most important functions of these ATPases are homeostasis, potassium regulation, and maintaining a gradient in different cell types, like epithelial cells. Na(+)/K(+)-ATPase has become a center of attention ever since it was proposed that it might play a crucial role in neurological disorders such as bipolar disorder, mania, depression, familial hemiplegic migraine, rapid-onset dystonia parkinsonism, chronic stress, epileptogenesis, and Alzheimer's disease. On the other hand, it has been reported that lithium could have a neuroprotective effect against ouabain, which is the best known Na(+)/K(+)-ATPase inhibitor, but and high concentrations of lithium could affect negatively H(+)/K(+)-ATPase activity, that has a key role in regulating acidosis and potassium deficiencies. Finally, potassium homeostasis regulation is composed of two main mechanisms, extrarenal and renal. Extrarenal mechanism controls plasma levels, shifting potassium from the extracellular to the intracellular, whereas renal mechanism concerns with body balance and is influenced by potassium intake and its urinary excretion. In this article, we discuss the functions, isoforms, and localization of P2C-type ATPases, describe some of their modulators, and discuss their implications in some diseases. PMID:25631710

  3. cAMP can raise or lower cardiac actomyosin ATPase activity depending on alpha-adrenergic activity.

    PubMed

    McClellan, G; Weisberg, A; Winegrad, S

    1994-08-01

    Adenosine 3',5'-cyclic monophosphate (cAMP) or beta-adrenergic stimulation has been shown to increase actomyosin adenosinetriphosphatase (ATPase) activity in cardiac muscle. Because the major catecholamine transmitters have both alpha- and beta-adrenergic activity, the possibility of a role for alpha-adrenergic stimulation in the regulation of ATPase activity has been investigated. Histochemical measurement of actomyosin ATPase activity in quickly frozen rat hearts has been used as the assay of enzymatic function of the contractile proteins. The dose-response curve of ATPase activity to cAMP shows an increase in ATPase activity at a threshold concentration of 0.01 microM, a peak effect at 0.5-1.0 microM, and a decline beyond 1.5 microM to a level below control at 10 microM cAMP. Kinetic studies varying ATP concentration from 0.5 to 10 mM indicated the existence of multiple forms of actomyosin ATPase activity in the absence of cAMP and only one form with a higher maximum velocity in the presence of 1 microM cAMP. Apparently cAMP raises the enzymatic activity of the individual actomyosin molecule rather than increasing the number of active molecules. The addition of an alpha-adrenergic blocker had no significant effect in the absence of added cAMP, but in the presence of the cyclic nucleotide, 1 microM prazosin always produced a negative effect on ATPase activity. Over the entire range of 0.01-10 microM, cAMP lowered ATPase activity when the alpha-adrenergic antagonist was present. The integrity of the cAMP regulatory system was sensitive to the tissue oxygen tension at the time the heart was quickly frozen. At certain oxygen tension, the stimulatory component of the cAMP regulation was observed without any inhibitory component, suggesting that there are two relatively independent parts of the regulatory mechanism, an inhibitory and a stimulatory. In the presence of gamma-labeled [32P]ATP, 32P was incorporated into several proteins, including the inhibitory subunit of troponin (TNI), C protein, and the regulatory light chain of myosin. cAMP (1 microM) caused an increase in 32P labeling of TNI and C protein. The addition of prazosin with cAMP caused a decrease in the overall level of phosphorylation with specific dephosphorylation of C protein and TNI, the former to a degree similar to the decrease in actomyosin ATPase activity, the latter to a greater degree. These results indicate that alpha-adrenergic activity modulates the balance between kinase and phosphatase activity in the presence of cAMP, probably by inhibiting phosphatase activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7915082

  4. Computational Classification of P-Type ATPases.

    PubMed

    Søndergaard, Dan; Knudsen, Michael; Pedersen, Christian Nørgaard Storm

    2016-01-01

    Analysis of sequence data is inevitable in modern molecular biology, and important information about for example proteins can be inferred efficiently using computational methods. Here, we explain how to use the information in freely available databases together with computational methods for classification and motif detection to assess whether a protein sequence corresponds to a P-type ATPase (and if so, which subtype) or not. PMID:26695056

  5. Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity

    PubMed Central

    Sanborn, Keri B.; Mace, Emily M.; Rak, Gregory D.; Difeo, Analisa; Martignetti, John A.; Pecci, Alessandro; Bussel, James B.; Favier, Rémi

    2011-01-01

    Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity. PMID:22123909

  6. Identification and localization of myosin phosphatase in human platelets.

    PubMed Central

    Murányi, A; Erdodi, F; Ito, M; Gergely, P; Hartshorne, D J

    1998-01-01

    Type 1 (PP1) and type 2A (PP2A) phosphatase activity was measured in three subcellular fractions of human platelets. About 80% of the activity was in the high-speed supernatant. Western blots showed that the catalytic subunit of PP1 (PP1c), including alpha- and delta-isoforms, was present in each fraction, but the level of the catalytic subunit of PP2A was very low in the low-speed pellet (cytoskeletal fraction). Various antibodies detected a subunit similar to the 130 kDa subunit (M130) of myosin phosphatase (MP) of smooth muscle in the low- and the high-speed pellets of human platelets. PP1c and associated proteins were isolated by microcystin-Sepharose. Many proteins were separated from each fraction, including myosin, actin and PP1c. M130 was separated only from the low-speed and the high-speed pellets. Kinase activities were detected in the unbound fractions, and fractions from the low- and high-speed pellets phosphorylated M130 and myosin respectively. Treatment of platelets with calyculin A increased the phosphorylation level of many proteins, including myosin heavy- and light-chains, and caused association of cytoskeletal proteins with the low-speed pellet. No marked change in the distribution of PP1c and M130 was detected. These results suggest that the MP in human platelets is composed of PP1c plus a subunit similar to M130 of the smooth muscle phosphatase. PMID:9461514

  7. 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

  8. Rotational model for actin filament alignment by myosin.

    PubMed

    Miller, Callie J; Bard Ermentrout, G; Davidson, Lance A

    2012-05-01

    Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allows us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment which occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors. PMID:22326473

  9. Force generation by kinesin and myosin cytoskeletal motor proteins.

    PubMed

    Kull, F Jon; Endow, Sharyn A

    2013-01-01

    Kinesins and myosins hydrolyze ATP, producing force that drives spindle assembly, vesicle transport and muscle contraction. How do motors do this? Here we discuss mechanisms of motor force transduction, based on their mechanochemical cycles and conformational changes observed in crystal structures. Distortion or twisting of the central β-sheet - proposed to trigger actin-induced Pi and ADP release by myosin, and microtubule-induced ADP release by kinesins - is shown in a movie depicting the transition between myosin ATP-like and nucleotide-free states. Structural changes in the switch I region form a tube that governs ATP hydrolysis and Pi release by the motors, explaining the essential role of switch I in hydrolysis. Comparison of the motor power strokes reveals that each stroke begins with the force-amplifying structure oriented opposite to the direction of rotation or swing. Motors undergo changes in their mechanochemical cycles in response to small-molecule inhibitors, several of which bind to kinesins by induced fit, trapping the motors in a state that resembles a force-producing conformation. An unusual motor activator specifically increases mechanical output by cardiac myosin, potentially providing valuable information about its mechanism of function. Further study is essential to understand motor mechanochemical coupling and energy transduction, and could lead to new therapies to treat human disease. PMID:23487037

  10. 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

  11. Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens

    PubMed Central

    Lutz, Gordon J; Cuizon, Denise B; Ryan, Allen F; Lieber, Richard L

    1998-01-01

    Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS-PAGE and only one adult hindlimb MHC isoform has been cloned. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Ranapipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS-PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co-migrated with type 1 MHC. The combined data from immunohistochemistry and SDS-PAGE suggests that Rana fibre types are composed of four different MHCs. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno-identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation. PMID:9518724

  12. Kinesin ATPase: Rate-limiting ADP release

    SciTech Connect

    Hackney, D.D.

    1988-09-01

    The ATPase rate of kinesin isolated from bovine brain by the method of S.A. Kuznetsov and V.I. Gelfand is stimulated 1000-fold by interaction with tubulin. The tubulin-stimulated reaction exhibits no extra incorporation of water-derived oxygens over a wide range of ATP and tubulin concentrations, indicating that P/sub i/ release is faster than the reversal of hydrolysis. ADP release, however, is slow for the basal reaction and its release is rate limiting as indicated by the very tight ADP binding (K/sub i/ < 5 nM), the retention of a stoichiometric level of bound ADP through ion-exchange chromatography and dialysis, and the reversible labeling of a bound ADP by (/sup 14/C)ATP at the steady-state ATPase rate as shown by centrifuge gel filtration and inaccessibility to pyruvate kinase. Tubulin accelerates the release of the bound ADP consistent with its activation of the net ATPase reaction. The detailed kinetics of ADP release in the presence of tubulin are biphasic indicating apparent heterogeneity with a fraction of the kinesin active sites being unaffected by tubulin.

  13. Myosin superfamily: The multi-functional and irreplaceable factors in spermatogenesis and testicular tumors.

    PubMed

    Li, Yan-Ruide; Yang, Wan-Xi

    2016-01-15

    Spermatogenesis is a fundamental process in sexual development and reproduction, in which the diploid spermatogonia transform into haploid mature spermatozoa. This process is under the regulation of multiple factors and pathway. Myosin has been implicated in various aspects during spermatogenesis. Myosins constitute a diverse superfamily of actin-based molecular motors that translocate along microfilament in an ATP-dependent manner, and six kinds of myosins have been proved that function during spermatogenesis. In mitosis and meiosis, myosins play an important role in spindle assembly and positioning, karyokinesis and cytokinesis. During spermiogenesis, myosins participate in acrosomal formation, nuclear morphogenesis, mitochondrial translocation and spermatid individualization. In this review, we summarize current understanding of the functions of myosin in spermatogenesis and some reproductive system diseases such as testicular tumors and prostate cancer, and discuss the roles of possible upstream molecules which regulate myosin in these processes. PMID:26478466

  14. Electrophoretic pattern, thermal denaturation, and in vitro digestibility of oxidized myosin.

    PubMed

    Liu, G; Xiong, Y L

    2000-03-01

    Physicochemical changes and in vitro digestibility of chicken breast myosin oxidized with a nonenzymic free-radical-generating system (FeCl(3)/H(2)O(2)/ascorbate) were studied by SDS-PAGE, differential scanning calorimetry, and o-phthaldialdehyde assay. Oxidation caused fragmentation and polymerization of myosin. Myosin polymers were cross-linked mainly through disulfide bonds. Hydroxyl radicals destabilized myosin, lowering its denaturation temperature by up to 4 degrees C. Oxidized myosin also produced a new thermal transition in the 60-80 degrees C temperature range, which could be attributed to the formation of disulfide-stabilized polymers. The proteolytic susceptibility of myosin to pepsin, trypsin, and chymotrypsin was increased by oxidation. Under nonreducing conditions, however, oxidized myosin showed decreased digestibility. The results may help explain variations in the functionality and nutritional quality of muscle foods in meat processing in which oxidation is involved. PMID:10725125

  15. 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. Conclusion Dictyostelium contains thirteen myosins together with 6–8 MLCs (myosin light chain) to assist in a variety of actin-based processes in the cell. Although they are homologous to myosins of higher eukaryotes, the myosins of Dictyostelium should be considered with care as models for specific functions of vertebrate myosins. PMID:16857047

  16. Earning stripes: myosin binding protein-C interactions with actin.

    PubMed

    van Dijk, Sabine J; Bezold, Kristina L; Harris, Samantha P

    2014-03-01

    Myosin binding protein-C (MyBP-C) was first discovered as an impurity during the purification of myosin from skeletal muscle. However, soon after its discovery, MyBP-C was also shown to bind actin. While the unique functional implications for a protein that could cross-link thick and thin filaments together were immediately recognized, most early research nonetheless focused on interactions of MyBP-C with the thick filament. This was in part because interactions of MyBP-C with the thick filament could adequately explain most (but not all) effects of MyBP-C on actomyosin interactions and in part because the specificity of actin binding was uncertain. However, numerous recent studies have now established that MyBP-C can indeed bind to actin through multiple binding sites, some of which are highly specific. Many of these interactions involve critical regulatory domains of MyBP-C that are also reported to interact with myosin. Here we review current evidence supporting MyBP-C interactions with actin and discuss these findings in terms of their ability to account for the functional effects of MyBP-C. We conclude that the influence of MyBP-C on muscle contraction can be explained equally well by interactions with actin as by interactions with myosin. However, because data showing that MyBP-C binds to either myosin or actin has come almost exclusively from in vitro biochemical studies, the challenge for future studies is to define which binding partner(s) MyBP-C interacts with in vivo. PMID:24442149

  17. 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. PMID:25733667

  18. Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats.

    PubMed

    Karim, Kamarulzaman; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-04-01

    We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17β (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17β followed by 3 days vehicle, progesterone, or estradiol-17β plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence. PMID:26403527

  19. Voltage Clamp Fluorometry of P-Type ATPases.

    PubMed

    Dempski, Robert E

    2016-01-01

    Voltage clamp fluorometry has become a powerful tool to compare partial reactions of P-type ATPases such as the Na(+),K(+)-ATPase and H(+),K(+)-ATPase with conformational dynamics of these ion pumps. Here, we describe the methodology to heterologously express membrane proteins in X. laevis oocytes and site-specifically label these proteins with one or more fluorophores. Fluorescence changes are measured simultaneously with current measurements under two-electrode voltage clamp conditions. PMID:26695040

  20. The 7-stranded structure of relaxed scallop muscle myosin filaments: support for a common head configuration in myosin-regulated muscles.

    PubMed

    Al-Khayat, Hind A; Morris, Edward P; Squire, John M

    2009-05-01

    Isolated relaxed myosin filaments from the myosin-regulated scallop striated adductor muscle have been reconstructed using electron microscopy and single particle analysis of negatively stained filaments. Three-dimensional reconstruction using 7-fold rotational symmetry but without imposed helical symmetry confirmed that the myosin head array is a 7-stranded, right-handed long-pitch 24/1 helix (or left-handed short-pitch 10/1 helix) with the whole structure having an axial repeat of 1440A. Reconstruction using the full helical symmetry revealed details of the myosin head density distribution within the head crowns in the relaxed scallop myosin filament. The resulting density distribution can best be explained by an arrangement in which the two heads from the same myosin molecule interact together within each crown in a compact parallel fashion along the filament axis. The configuration is consistent with the published configuration of the two heads within vertebrate smooth muscle myosin molecules observed in two-dimensional crystals of smooth muscle myosin and in the structure of tarantula myosin filaments. All these three muscle types are myosin-regulated, providing further support for a general motif of intramolecular interacting-heads structure in the relaxed state of myosin-regulated muscles as was proposed earlier by Woodhead et al. [Woodhead, J.L., Zhao, F.-Q., Craig, R., Egelman, E.H., Alamo, L., Padron, R.. 2005. Atomic model of a myosin filament in the relaxed state. Nature 436, 1195-1199]. However, the orientation of the Wendt structure is different from that found by Woodhead in that the outer head projects outwards and the inner head lies closer to the filament backbone, as in earlier work done on the insect flight muscle myosin filaments [AL-Khayat, H.A., Hudson, L., Reedy, M.K., Irving, T.C., Squire, J.M., 2003. Myosin head configuration in relaxed insect flight muscle: X-ray modelled resting crossbridges in a pre-powerstroke state are poised for actin binding. Biophys. J. 85, 1063-1079]. Possible species specific details that may differ between the scallop and the tarantula myosin filaments are also discussed. PMID:19248832

  1. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium.

    PubMed Central

    Lowes, B D; Minobe, W; Abraham, W T; Rizeq, M N; Bohlmeyer, T J; Quaife, R A; Roden, R L; Dutcher, D L; Robertson, A D; Voelkel, N F; Badesch, D B; Groves, B M; Gilbert, E M; Bristow, M R

    1997-01-01

    Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction. PMID:9410910

  2. Changes in gene expression in the intact human heart. Downregulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium.

    PubMed

    Lowes, B D; Minobe, W; Abraham, W T; Rizeq, M N; Bohlmeyer, T J; Quaife, R A; Roden, R L; Dutcher, D L; Robertson, A D; Voelkel, N F; Badesch, D B; Groves, B M; Gilbert, E M; Bristow, M R

    1997-11-01

    Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction. PMID:9410910

  3. Solution structure of myosin-ADP-MgFn ternary complex by fluorescent probes and small-angle synchrotron X-ray scattering.

    PubMed

    Maruta, S; Aihara, T; Uyehara, Y; Homma, K; Sugimoto, Y; Wakabayashi, K

    2000-10-01

    In the presence of excess amounts of fluorine, a physiological divalent cation, magnesium (Mg(2+)), forms a novel phosphate analogue, magnesium fluoride (MgFn). Park et al. [Biochim. Biophys. Acta 1430, 127-140 (1999)] previously demonstrated that MgADP. MgFn forms a complex with myosin subfragment-1 (S-1), and the S-1.ADP. MgFn ternary complex mimics a transient state in the activity cycle of ATPase. In the present study, localized conformations in the regions of highly reactive cysteine and lysine residues, Cys 707 (SH1), Cys 697 (SH2), and Lys 83 (RLR), which change their conformations markedly during ATP hydrolysis, were studied using fluorescent probes and chemical modification. The global shape of the complex was also studied using small angle X-ray solution scattering and compared it with other previously reported myosin.ADP. fluorometal ternary complexes. The results suggest that the overall conformation and localized functional regions of the complex are quite similar to those in the presence of ATP, indicating that the complex mimics the M(**).ADP.P(i) steady state. PMID:11011152

  4. cGMP-dependent protein kinase Iβ regulates breast cancer cell migration and invasion via interaction with the actin/myosin-associated protein caldesmon

    PubMed Central

    Schwappacher, Raphaela; Rangaswami, Hema; Su-Yuo, Jacqueline; Hassad, Aaron; Spitler, Ryan; Casteel, Darren E.

    2013-01-01

    Summary The two isoforms of type I cGMP-dependent protein kinase (PKGIα and PKGIβ) differ in their first ∼100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein–protein interactions. Using the PKGIβ dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGIβ-specific binding protein. PKGIβ phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGIβ and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGIβ enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGIβ in human breast cancer cells, suggesting that PKGIβ is a potential target for breast cancer treatment. PMID:23418348

  5. The RhoA-Rok-Myosin II Pathway is Involved in Extracellular Matrix-Mediated Regulation of Prolactin Signaling in Mammary Epithelial Cells

    PubMed Central

    Du, Jyun-Yi; Chen, Meng-Chi; Hsu, Tsai-Ching; Wang, Jen-Hsing; Brackenbury, Lisa; Lin, Ting-Hui; Wu, Yi-Ying; Yang, Zhihong; Streuli, Charles H; Lee, Yi-Ju

    2012-01-01

    In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell–BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and β-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell–matrix interactions. J. Cell. Physiol. 227: 1553–1560, 2012. © 2011 Wiley Periodicals, Inc. PMID:21678418

  6. Transcriptional regulators of Na,K-ATPase subunits

    PubMed Central

    Li, Zhiqin; Langhans, Sigrid A.

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

  7. 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

  8. Conformational changes of the myosin heads during hydrolysis of ATP as analyzed by x-ray solution scattering.

    PubMed

    Sugimoto, Y; Tokunaga, M; Takezawa, Y; Ikebe, M; Wakabayashi, K

    1995-04-01

    We have shown for the first time that the myosin head (subfragment-1, S1), the energy-transducing component in the actomyosin motor system undergoes a distinct shape change during hydrolysis of ATP using x-ray solution scattering techniques. Among various analogs for intermediate states of the S1 ATPase cycle, the complexes with MgADP and vanadate (S1.ADP.Vi), MgADP and beryllium fluoride (S1.ADP.BeF3), or MgADP and aluminum fluoride (S1.ADP.AIF4) showed a shape change similar to that in the presence of MgATP, but the complexes with ATP gamma S (S1.ADP gamma S) and MgADP trapped by cross-linking with pPDM (S1.ADP-pPDM) seemed to have a shape similar to that of nucleotide-free S1. These results indicate that the shape of an S1**.ADP.Pi state is more rounded or bent than in other intermediate states of the S1 ATPase cycle. Such changes occur in light chain 2-deficient S1 and also in smooth muscle S1. However, MgADP-fluoride complexes with smooth muscle S1 (without phosphorylation of a regulatory light chain) seemed to have a structure similar to that of nucleotide-free S1. Analysis of x-ray scattering data indicated that a conformational change of S1 in the presence of MgATP might be caused by a hinge-like bending movement between the catalytic and regulatory domains. The global change of S1 is correlated with some specific changes of a nucleotide-binding moiety. PMID:7787093

  9. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

    PubMed

    Koenderink, Jan B; Swarts, Herman G P

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system. PMID:26695023

  10. Na(+),K (+)-ATPase as a docking station: protein-protein complexes of the Na(+),K (+)-ATPase.

    PubMed

    Reinhard, Linda; Tidow, Henning; Clausen, Michael J; Nissen, Poul

    2013-01-01

    The Na(+),K(+)-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na(+) ions out of the cell and of K(+) ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na(+),K(+)-ATPase, recent work has suggested additional roles for Na(+),K(+)-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na(+),K(+)-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na(+),K(+)-ATPase as a signal transducer, but also briefly discuss other Na(+),K(+)-ATPase protein-protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme. PMID:22695678

  11. Three-dimensional structure of the human myosin thick filament: clinical implications

    PubMed Central

    AL-Khayat, Hind A.

    2013-01-01

    High resolution information about the three-dimensional (3D) structure of myosin filaments has always been hard to obtain. Solving the 3D structure of myosin filaments is very important because mutations in human cardiac muscle myosin and its associated proteins (e.g. titin and myosin binding protein C) are known to be associated with a number of familial human cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand how normal heart muscle works and how it fails, as well as the effects of the known mutations on muscle contractility, it is essential to properly understand myosin filament 3D structure and properties in both healthy and diseased hearts. The aim of this review is firstly to provide a general overview of the 3D structure of myosin thick filaments, as studied so far in both vertebrates and invertebrate striated muscles. Knowledge of this 3D structure is the starting point from which myosin filaments isolated from human cardiomyopathic samples, with known mutations in either myosin or its associated proteins (titin or C-protein), can be studied in detail. This should, in turn, enable us to relate the structure of myosin thick filament to its function and to understanding the disease process. A long term objective of this research would be to assist the design of possible therapeutic solutions to genetic myosin-related human cardiomyopathies. PMID:24689030

  12. A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

    PubMed Central

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R.

    2006-01-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus. PMID:16837551

  13. Topography of a vacuolar-type H+-translocating ATPase: chromaffin-granule membrane ATPase I.

    PubMed Central

    Apps, D K; Percy, J M; Perez-Castineira, J R

    1989-01-01

    Proteins exposed on the cytoplasmic face of isolated chromaffin granules were labelled by lactoperoxidase-catalysed radioiodination and by non-enzymic biotinylation. Granule membranes were then prepared, and the H+-translocating ATPase isolated by fractionation with Triton X-114. The labelling of individual ATPase subunits was assessed by polyacrylamide-gel electrophoresis, followed by autoradiography or by blotting and decoration with 125I-labelled streptavidin. Subunits of 72, 57 and kDa were strongly labelled, and could be removed from the membrane at pH 11: they are therefore extrinsic proteins. The 120 kDa subunit was also labelled, but it was not solubilized at pH 11. Photolabelling with a hydrophobic probe indicated that this subunit penetrates the bilayer, and enzymic degradation studies showed the presence of N-linked oligosaccharides; this subunit therefore spans the chromaffin-granule membrane. Labelling of the 17 kDa subunit occurred predominantly on the extracytoplasmic (matrix) face of the granule membrane. These results are consistent with this V-type ATPase having a structure that is generally similar to that of mitochondrial (F-type) ATPases, although the attachment of the 120 kDa subunit may be asymmetrical. Images Fig. 1. Fig. 2. Fig. 5. PMID:2532503

  14. Eukaryotic V-ATPase: novel structural findings and functional insights.

    PubMed

    Marshansky, Vladimir; Rubinstein, John L; Grüber, Gerhard

    2014-06-01

    The eukaryotic V-type adenosine triphosphatase (V-ATPase) is a multi-subunit membrane protein complex that is evolutionarily related to F-type adenosine triphosphate (ATP) synthases and A-ATP synthases. These ATPases/ATP synthases are functionally conserved and operate as rotary proton-pumping nano-motors, invented by Nature billions of years ago. In the first part of this review we will focus on recent structural findings of eukaryotic V-ATPases and discuss the role of different subunits in the function of the V-ATPase holocomplex. Despite structural and functional similarities between rotary ATPases, the eukaryotic V-ATPases are the most complex enzymes that have acquired some unconventional cellular functions during evolution. In particular, the novel roles of V-ATPases in the regulation of cellular receptors and their trafficking via endocytotic and exocytotic pathways were recently uncovered. In the second part of this review we will discuss these unique roles of V-ATPases in modulation of function of cellular receptors, involved in the development and progression of diseases such as cancer and diabetes as well as neurodegenerative and kidney disorders. Moreover, it was recently revealed that the V-ATPase itself functions as an evolutionarily conserved pH sensor and receptor for cytohesin-2/Arf-family GTP-binding proteins. Thus, in the third part of the review we will evaluate the structural basis for and functional insights into this novel concept, followed by the analysis of the potentially essential role of V-ATPase in the regulation of this signaling pathway in health and disease. Finally, future prospects for structural and functional studies of the eukaryotic V-ATPase will be discussed. PMID:24508215

  15. Structural aspects of proton-pumping ATPases.

    PubMed

    Walker, J E; Fearnley, I M; Lutter, R; Todd, R J; Runswick, M J

    1990-01-30

    ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of alpha 3 beta 3 gamma 1 delta 1 epsilon 1 a1b2c10-12. The first five of these constitute a globular structure, F1-ATPase, which is bound to an intrinsic membrane domain, F0, an assembly of the three remaining subunits. ATP synthases driven by photosynthesis are slightly more complex. In chloroplasts, and probably in photosynthetic bacteria, they have nine subunits, all homologues of the components of the E. coli enzyme; the additional subunit is a duplicated and diverged relation of subunit b. The mammalian mitochondrial enzyme is more complex. It contains 14 different polypeptides, of which 13 have been characterized. Two membrane components, a (or ATPase-6) and A6L, are encoded in the mitochondrial genome in overlapping genes and the remaining subunits are nuclear gene products that are translated on cytoplasmic ribosomes and then imported into the organelle. The sequence of the proteins of ATP-synthase have provided information about amino acids that are important for its function. For example, amino acids contributing to nucleotide binding sites have been identified. Also, they provide the basis of models of secondary structure of membrane components that constitute the transmembrane proton channel. An understanding of the coupling of the transmembrane potential gradient for protons, delta mu H+, to ATP synthesis will probably require the determination of the structure of the entire membrane bound complex. Crystals have been obtained of the globular domain, F1-ATPase. They diffract to a resolution of 3-4 A and data collection is in progress. As a preliminary step towards crystallization of the entire complex, we have purified it from bovine mitochondria and reconstituted it into phospholipid vesicles. PMID:1970643

  16. AZT binding to Na,K-ATPase.

    PubMed

    Ahmed-Ouameur, A; Neault, J-F; Claveau, S; Tajmir-Riahi, H A

    2005-01-01

    3'-azido-3'-deoxythymidine (AZT) is the first effective drug used clinically for the treatment of human immunodeficiency virus (HIV) infection. The drug interactions with DNA and protein are associated with its mechanism of action in vivo. This study was designed to examine the interaction of AZT with the Na,K-dependent adenosine triphosphatase (Na,K-ATPase) in H2O and D2O solutions at physiological pH using drug concentration of 0.1 microM to 1 mM and final protein concentration of 0.5 to 1 mg/mL. Ultraviolet absorption and Fourier transform infrared difference spectroscopy with its self-deconvolution, second-derivative resolution enhancement, and curve-fitting procedures were used to characterize the drug-binding mode, the drug-binding constant, and the effects of drug interaction on the protein secondary structure. Spectroscopic evidence showed that at low drug concentration (0.1 microM), AZT binds (H-bonding) mainly to the polypeptide C=O and C-N groups with two binding constants of K1 = 5.3 x 10(5) M(-1) and K2 = 9.8 x 10(3) M(-1). As drug content increased, AZT-lipid complex prevailed. At a high drug concentration (1 mM), drug binding resulted in minor protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38%, in the free Na,K-ATPase to that of the alpha-helix 19%; beta-pleated 21.1%; turn 10.1%; beta-antiparallel 8.8% and random 41%, in the AZT-ATPase complexes. PMID:15673931

  17. Stochastic Force Generation by Small Ensembles of Myosin II Motors

    NASA Astrophysics Data System (ADS)

    Erdmann, Thorsten; Schwarz, Ulrich S.

    2012-05-01

    Forces in the actin cytoskeleton are generated by small groups of nonprocessive myosin II motors for which stochastic effects are highly relevant. Using a cross-bridge model with the assumptions of fast power-stroke kinetics and equal load sharing between equivalent states, we derive a one-step master equation for the activity of a finite-sized ensemble of mechanically coupled myosin II motors. For constant external load, this approach yields analytical results for duty ratio and force-velocity relation as a function of ensemble size. We find that stochastic effects cannot be neglected for ensemble sizes below 15. The one-step master equation can be used also for efficient computer simulations with linear elastic external load and reveals the sequence of buildup of force and ensemble rupture that is characteristic for reconstituted actomyosin contractility.

  18. 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.

  19. Purification and Properties of an ATPase from Sulfolobus solfataricus

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Stan-Lotter, Helga

    1992-01-01

    A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzo-furazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-CL was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuri-phenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethyimaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

  20. Function, structure and regulation of the vacuolar (H+)-ATPases

    PubMed Central

    Jefferies, Kevin C.; Cipriano, Daniel J.; Forgac, Michael

    2008-01-01

    The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V1) responsible for hydrolysis of ATP and an integral domain (V0) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer. PMID:18406336

  1. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation.

    PubMed

    Rozbicki, Emil; Chuai, Manli; Karjalainen, Antti I; Song, Feifei; Sang, Helen M; Martin, René; Knölker, Hans-Joachim; MacDonald, Michael P; Weijer, Cornelis J

    2015-04-01

    Primitive streak formation in the chick embryo involves large-scale highly coordinated flows of more than 100,000 cells in the epiblast. These large-scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combination of light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression, and asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localization and direction of which correlate strongly with the appearance of active myosin II cables in aligned apical junctions in neighbouring cells. Use of class specific myosin inhibitors and gene-specific knockdown shows that apical contraction and intercalation are myosin II dependent and also reveal critical roles for myosin I and myosin V family members in the assembly of junctional myosin II cables. PMID:25812521

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

    PubMed Central

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

    2013-01-01

    Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca2+-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI–specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane. PMID:23382463

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

    PubMed

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

    2013-02-01

    Before undergoing neuroexocytosis, secretory granules (SGs) are mobilized and tethered to the cortical actin network by an unknown mechanism. Using an SG pull-down assay and mass spectrometry, we found that myosin VI was recruited to SGs in a Ca(2+)-dependent manner. Interfering with myosin VI function in PC12 cells reduced the density of SGs near the plasma membrane without affecting their biogenesis. Myosin VI knockdown selectively impaired a late phase of exocytosis, consistent with a replenishment defect. This exocytic defect was selectively rescued by expression of the myosin VI small insert (SI) isoform, which efficiently tethered SGs to the cortical actin network. These myosin VI SI-specific effects were prevented by deletion of a c-Src kinase phosphorylation DYD motif, identified in silico. Myosin VI SI thus recruits SGs to the cortical actin network, potentially via c-Src phosphorylation, thereby maintaining an active pool of SGs near the plasma membrane. PMID:23382463

  4. Role of plant myosins in motile organelles: is a direct interaction required?

    PubMed

    Buchnik, Limor; Abu-Abied, Mohamad; Sadot, Einat

    2015-01-01

    Plant organelles are highly motile, with speed values of 3-7 µm/s in cells of land plants and about 20-60 µm/s in characean algal cells. This movement is believed to be important for rapid distribution of materials around the cell, for the plant's ability to respond to environmental biotic and abiotic signals and for proper growth. The main machinery that propels motility of organelles within plant cells is based on the actin cytoskeleton and its motor proteins the myosins. Most plants express multiple members of two main classes: myosin VIII and myosin XI. While myosin VIII has been characterized as a slow motor protein, myosins from class XI were found to be the fastest motor proteins known in all kingdoms. Paradoxically, while it was found that myosins from class XI regulate most organelle movement, it is not quite clear how or even if these motor proteins attach to the organelles whose movement they regulate. PMID:25196231

  5. Non-muscle myosins in tumor progression, cancer cell invasion and metastasis

    PubMed Central

    Ouderkirk, J. L.; Krendel, M.

    2014-01-01

    The actin cytoskeleton, which regulates cell polarity, adhesion, and migration, can influence cancer progression, including initial acquisition of malignant properties by normal cells, invasion of adjacent tissues, and metastasis to distant sites. Actin-dependent molecular motors, myosins, play key roles in regulating tumor progression and metastasis. In this review, we examine how non-muscle myosins regulate neoplastic transformation and cancer cell migration and invasion. Members of the myosin superfamily can act as either enhancers or suppressors of tumor progression. This review summarizes the current state of knowledge on how mutations or epigenetic changes in myosin genes and changes in myosin expression may affect tumor progression and patient outcomes and discusses the proposed mechanisms linking myosin inactivation or upregulation to malignant phenotype, cancer cell migration, and metastasis. PMID:25087729

  6. Myosin II-mediated cell shape changes and cell intercalation contribute to primitive streak formation

    PubMed Central

    Song, Feifei; Sang, Helen M.; Martin, René; Knölker, Hans-Joachim; MacDonald, Michael P; Weijer, Cornelis J

    2016-01-01

    Primitive streak formation in the chick embryo involves large scale highly coordinated flows of over 100.000 cells in the epiblast. These large scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combined light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression as well as asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localisation and direction of which correlate strongly with the appearance of active Myosin II cables in aligned apical junctions in neighbouring cells. Use of a class specific Myosin inhibitors and gene specific knockdowns show that apical contraction and intercalation are Myosin II dependent and also reveal critical roles for Myosin I and Myosin V family members in the assembly of junctional Myosin II cables. PMID:25812521

  7. Myosin Vs organize actin cables in fission yeast

    PubMed Central

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

    2012-01-01

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

  8. Myosin VI contributes to malignant proliferation of human glioma cells

    PubMed Central

    Xu, Rong; Fang, Xu-hao

    2016-01-01

    Previously characterized as a backward motor, myosin VI (MYO6), which belongs to myosin family, moves toward the minus end of the actin track, a direction opposite to all other known myosin members. Recent researches have illuminated the role of MYO6 in human cancers, particularly in prostate cancer. However, the role of MYO6 in glioma has not yet been determined. In this study, to explore the role of MYO6 in human glioma, lentivirus-delivered short hairpin RNA (shRNA) targeting MYO6 was designed to stably down-regulate its endogenous expression in glioblastoma cells U251. Knockdown of MYO6 signifi cantly inhibited viability and proliferation of U251 cells in vitro. Moreover, the cell cycle of U251 cells was arrested at G0/G1 phase with the absence of MYO6, which could contribute to the suppression of cell proliferation. In conclusion, we firstly identified the crucial involvement of MYO6 in human glioma. The inhibition of MYO6 by shRNA might be a potential therapeutic method in human glioma. PMID:26937209

  9. Actin-myosin viscoelastic flow in the keratocyte lamellipod.

    PubMed

    Rubinstein, Boris; Fournier, Maxime F; Jacobson, Ken; Verkhovsky, Alexander B; Mogilner, Alex

    2009-10-01

    The lamellipod, the locomotory region of migratory cells, is shaped by the balance of protrusion and contraction. The latter is the result of myosin-generated centripetal flow of the viscoelastic actin network. Recently, quantitative flow data was obtained, yet there is no detailed theory explaining the flow in a realistic geometry. We introduce models of viscoelastic actin mechanics and myosin transport and solve the model equations numerically for the flat, fan-shaped lamellipodial domain of keratocytes. The solutions demonstrate that in the rapidly crawling cell, myosin concentrates at the rear boundary and pulls the actin network inward, so the centripetal actin flow is very slow at the front, and faster at the rear and at the sides. The computed flow and respective traction forces compare well with the experimental data. We also calculate the graded protrusion at the cell boundary necessary to maintain the cell shape and make a number of other testable predictions. We discuss model implications for the cell shape, speed, and bi-stability. PMID:19804715

  10. Structural determinants of cooperativity in acto-myosin interactions.

    PubMed

    Moraczewska, Joanna

    2002-01-01

    Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between neighboring tropomyosin molecules is not necessary for the cooperativity of the thin filament activation. When direct contacts between tropomyosin molecules are disrupted, the conformational changes in the filament are most probably transmitted cooperatively through actin subunits, although the exact nature of these changes is not known. The function of tropomyosin ends, alternatively expressed in various isoforms, is to confer specific actin affinity. Tropomyosin's affinity or actin is directly related to the size of the apparent cooperative unit defined as the number of actin subunits turned into the active state by binding of one myosin head. Inner sequences of tropomyosin, particularly actin-binding periods 3 to 5, play crucial role in myosin-induced activation of the thin filament. A plausible mechanism of tropomyosin function in this process is that inner tropomyosin regions are either specifically recognized by myosin or they define the right actin conformation required for tropomyosin movement from its blocking position. PMID:12545187

  11. ADP KINASE AND ATPASE IN CHLOROPLASTS*

    PubMed Central

    Lynn, W. S.; Straub, K. D.

    1969-01-01

    Treatment of chloroplasts with trypsin activates a light-requiring ATPase whose properties are strikingly similar to those of the light-requiring ADP kinase of chloroplasts. The observations here presented suggest that there exists, in chloroplasts, a reducible enzyme which, in its reduced state, catalyzes the reversible reaction: Pi-2 + ADP-3 + H+ ⇌ ATP-4 + H2O. By reduction and protonation of the catalytic site of this enzyme, light-driven electron flow in the chloroplast drives the reaction to the right. Hydrolysis of ATP proceeds only when the enzyme is reduced and when the proton concentration within the chloroplast is kept at low levels, viz., in the absence of light, in the presence of uncoupling agents which decrease the concentration of internal H+, or in the presence of electron acceptors which by oxidizing the internal electron acceptors also decrease the proton potential. Activation of the enzyme requires light; it remains active only in the presence of ATP. Hydrolysis of all the ATP results in inactivation of the ATPase. The membrane-bound protein CF2 limits the reversibility of the reaction by excluding ATP and H2O from the enzyme site. It also facilitates the ability of the chloroplasts to accumulate and to maintain high internal concentrations of such ions as ADP, Pi, PMS+, and imidazole. Images PMID:4240684

  12. P4 ATPases: Flippases in Health and Disease

    PubMed Central

    van der Mark, Vincent A.; Oude Elferink, Ronald P.J.; Paulusma, Coen C.

    2013-01-01

    P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed “lipid flipping”. Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse) of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role. PMID:23579954

  13. Purification and properties of an ATPase from Sulfolobus solfataricus

    NASA Technical Reports Server (NTRS)

    Hochstein, Lawrence I.; Stan-Lotter, Helga

    1992-01-01

    The paper reports properties of a sulfite-activated ATPase from Sulfolobus solfataricus, purified using ammonium sulfate precipitation, column chromatography on UltraGel and Sepharose 6B, and SDS-PAGE. The 92-fold purified enzyme had a relative molecular mass of 370,000. It could be dissociated into three subunits with respective molecular masses of 63,000, 48,000, and 24,000. The ATPase activity was found to be inhibitable by nitrate, N-ethylmaleimide (which bound predominantly to the largest subunit), and 4-chloro 7-nitrobenzofurazan, but not by azide, quercetin, or vanadate. While the ATPase from S. solfataricus shared a number of properties with the S. acidocaldarius ATPase, there were also significant differences suggesting the existence of several types of archaeal ATPases.

  14. Identification and characterization of Myosin from rat testicular peritubular myoid cells.

    PubMed

    Fernández, Dario; Bertoldi, Maria V; Gómez, Laura; Morales, Alfonsina; Callegari, Eduardo; Lopez, Luis A

    2008-12-01

    In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1. PMID:18716291

  15. Evolution and Classification of Myosins, a Paneukaryotic Whole-Genome Approach

    PubMed Central

    Sebé-Pedrós, Arnau; Grau-Bové, Xavier; Richards, Thomas A.; Ruiz-Trillo, Iñaki

    2014-01-01

    Myosins are key components of the eukaryotic cytoskeleton, providing motility for a broad diversity of cargoes. Therefore, understanding the origin and evolutionary history of myosin classes is crucial to address the evolution of eukaryote cell biology. Here, we revise the classification of myosins using an updated taxon sampling that includes newly or recently sequenced genomes and transcriptomes from key taxa. We performed a survey of eukaryotic genomes and phylogenetic analyses of the myosin gene family, reconstructing the myosin toolkit at different key nodes in the eukaryotic tree of life. We also identified the phylogenetic distribution of myosin diversity in terms of number of genes, associated protein domains and number of classes in each taxa. Our analyses show that new classes (i.e., paralogs) and domain architectures were continuously generated throughout eukaryote evolution, with a significant expansion of myosin abundance and domain architectural diversity at the stem of Holozoa, predating the origin of animal multicellularity. Indeed, single-celled holozoans have the most complex myosin complement among eukaryotes, with paralogs of most myosins previously considered animal specific. We recover a dynamic evolutionary history, with several lineage-specific expansions (e.g., the myosin III-like gene family diversification in choanoflagellates), convergence in protein domain architectures (e.g., fungal and animal chitin synthase myosins), and important secondary losses. Overall, our evolutionary scheme demonstrates that the ancestral eukaryote likely had a complex myosin repertoire that included six genes with different protein domain architectures. Finally, we provide an integrative and robust classification, useful for future genomic and functional studies on this crucial eukaryotic gene family. PMID:24443438

  16. Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A.

    PubMed

    Yang, Yidai; Ye, Qilu; Jia, Zongchao; Côté, Graham P

    2015-09-25

    The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min(-1), respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μM, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg(2+) ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3-6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PMID:26260792

  17. Molecular genetics of myosin motors in Arabidopsis. Progress report, [July 1, 1992--February 28, 1994

    SciTech Connect

    Not Available

    1994-06-01

    We have evidence for at least nine myosin-like genes in Arbidopsis, six of which have been cloned by a PCR-based method from genomic DNA, two have been isolated by genomic DNA cloning, and four have been identified by cDNA cloning. Most of our attention has been focused on the four myosin genes for which we have cDNA clones, and these cDNAs have now been sequenced to completion. Each of these myosins is similar in overall structure, with each containing the characteristic myosin head (motor) domain, which possesses ATP- and actin-binding motifs, a series of IQ repeats, which may be involved in calmodulin binding, a domain with a high probability of forming an alpha-helical coiled-coil secondary structure, which may allow the polypeptides to form dimers, and a variable tail domain, which may serve to define the specific cellular component that each myosin interacts with. One of these myosin genes, called MYA1, displays structural similarity to class of myosins that includes the yeast MYO2, mouse Dilute, and chicken p190 proteins, and this group of myosins is thought to play a role in intracellular trafficking of organelles. Because MYA1 is similar to this interesting class of myosins, we have chosen to conduct detailed studies of MYA1.

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

    NASA Astrophysics Data System (ADS)

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

    2004-01-01

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

  19. 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.

  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. Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation

    NASA Astrophysics Data System (ADS)

    Nucciotti, V.; Stringari, C.; Sacconi, L.; Vanzi, F.; Linari, M.; Piazzesi, G.; Lombardi, V.; Pavone, F. S.

    2009-02-01

    Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.

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

    PubMed Central

    Hariadi, R. F.; Sommese, R. F.; Adhikari, A. S.; Taylor, R. E.; Sutton, S.; Spudich, J. A.

    2016-01-01

    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 lattice1. 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 understood2. 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

  3. Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world.

    PubMed

    Kashiyama, T; Kimura, N; Mimura, T; Yamamoto, K

    2000-06-01

    In characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables. The speed of this sliding movement is the fastest among many molecular motors known so far. We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin. The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive alpha-helical coiled-coil domain, and a C-terminal globular domain. Phylogenetic analysis suggested that this myosin belongs to class XI. PMID:10833276

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

    PubMed Central

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

    2014-01-01

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

  5. Relationship between isometric force and myofibrillar MgATPase at short sarcomere length in skeletal and cardiac muscle and its relevance to the concept of activation heat.

    PubMed

    Stephenson, D George

    2003-08-01

    1. This paper has been written in recognition of the seminal contributions to cardiac and skeletal muscle energetics made by Professor Colin Gibbs during his distinguished academic career. 2. The paper focuses on what is now known about the relationship between Ca2+-activated isometric force production and myofibrillar MgATPase in intact and skinned (surface membrane rendered permeable) skeletal and cardiac muscle preparations at short sarcomere lengths. 3. The relevance of this relationship to understanding the interactions between the actin and myosin filaments at the cross-bridge level in the region of double actin filament overlap and the cellular basis of 'activation heat' measurements in intact striated muscles is discussed. PMID:12890181

  6. Use of Fluorescent Techniques to Study the In Vitro Movement of Myosins

    PubMed Central

    Toepfer, Christopher

    2014-01-01

    Myosins are a large superfamily of actin-dependent molecule motors that carry out many functions in cells. Some myosins are cargo carriers that move processively along actin which means that a single molecule of myosin can take many ATP-dependent steps on actin per initial encounter. Other myosins are designed to work in large ensembles such as myosin thick filaments. In vitro motility assays are a powerful method for studying the function of myosins. These assays in general use small amounts of protein, are simple to implement, and can be done on microscopes commonly found in many laboratories. There are two basic versions of the assay which involve different geometries. In the sliding actin in vitro motility assay, myosin molecules are bound to a coverslip surface in a simply constructed microscopic flow chamber. Fluorescently labeled actin filaments are added to the flow chamber in the presence of ATP, and the movement of these actin filaments powered by the surface-bound myosins is observed. This assay has been used widely for a variety of myosins including both processive and nonprocessive ones. From this assay, one can easily measure the rate at which myosin is translocating actin. The single-molecule motility assay uses an inverted geometry compared to the sliding actin in vitro motility assay. It is most useful for processive myosins. Here, actin filaments are affixed to the coverslip surface. Fluorescently labeled single molecules of myosins (usually ones with processive kinetics) are introduced, and the movement of single molecules along the actin filaments is observed. This assay typically uses total internal reflection fluorescent (TIRF) microscopy to reduce the background signal arising from myosins in solution. From this assay, one can measure the velocity of movement, the frequency of movement, and the run length. If sufficient photons can be collected, one can use Gaussian fitting of the point spread function to determine the position of the labeled myosin to within a few nanometers which allows for measurement of the step size and the stepping kinetics. Together, these two assays are powerful tools to elucidate myosin function. PMID:25095996

  7. Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP.

    PubMed

    Xia, Li; Yuwen, Liu; Jie, Li; Huilin, Li; Xi, Yang; Cunxin, Wang; Zhiyong, Wang

    2004-08-01

    Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM. PMID:15558949

  8. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    PubMed Central

    Mahmmoud, Yasser A.

    2008-01-01

    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K+. Studies with purified membranes revealed that CPZ reduced Na+-dependent phosphorylation by interference with Na+ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K+. Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive 22Na+ influx into K+-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na+ cycle from an Na+/K+ cycle in the pump. The Na+ cycle possibly involves transport through the recently characterized Na+-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na+ efflux by modifying the transport stoichiometry of single pump units. PMID:18230728

  9. Myosin 1E localizes to actin polymerization sites in lamellipodia, affecting actin dynamics and adhesion formation.

    PubMed

    Gupta, Prabuddha; Gauthier, Nils C; Cheng-Han, Yu; Zuanning, Yuan; Pontes, Bruno; Ohmstede, Malte; Martin, René; Knölker, Hans-Joachim; Döbereiner, Hans-Günther; Krendel, Mira; Sheetz, Michael

    2013-01-01

    Because the actin network in active lamellipodia is continuously assembling at the edge, moving inward and disassembling, there is a question as to how actin-binding proteins and other components are transported to the leading edge and how nascent adhesions are stabilized. Active transport could play a significant role in these functions but the components involved are unknown. We show here that Myosin 1E (a long tailed Myosin 1 isoform) rapidly moves to the tips of active lamellipodia and to actin-rich early adhesions, unlike Myosin 1G, 1B or 1C (short tailed isoforms). Myosin 1E co-localizes with CARMIL, FHOD1, Arp3 and β3-integrin in those early adhesions. But these structures precede stable paxillin-rich adhesions. Myosin 1E movement depends upon actin-binding domains and the presence of an SH3 oligomerization domain. Overexpression of a Myosin 1E deletion mutant without the extreme C-terminal interacting (SH3) domain (Myosin 1EΔSH3) increases edge fluctuations and decreases stable adhesion lifetimes. In contrast, overexpression of Myosin 1E full tail domain (TH1+TH2+TH3/SH3) decreases edge fluctuation. In Myosin 1E knockdown cells, and more prominently in cells treated with Myosin 1 inhibitor, cell-matrix adhesions are also short-lived and fail to mature. We suggest that, by moving to actin polymerization sites and early adhesion sites in active lamellipodia, Myosin 1E might play important roles in transporting not only important polymerizing proteins but also proteins involved in adhesion stabilization. PMID:24337113

  10. Myosin 1E localizes to actin polymerization sites in lamellipodia, affecting actin dynamics and adhesion formation

    PubMed Central

    Gupta, Prabuddha; Gauthier, Nils C.; Cheng-Han, Yu; Zuanning, Yuan; Pontes, Bruno; Ohmstede, Malte; Martin, René; Knölker, Hans-Joachim; Döbereiner, Hans-Günther; Krendel, Mira; Sheetz, Michael

    2013-01-01

    Summary Because the actin network in active lamellipodia is continuously assembling at the edge, moving inward and disassembling, there is a question as to how actin-binding proteins and other components are transported to the leading edge and how nascent adhesions are stabilized. Active transport could play a significant role in these functions but the components involved are unknown. We show here that Myosin 1E (a long tailed Myosin 1 isoform) rapidly moves to the tips of active lamellipodia and to actin-rich early adhesions, unlike Myosin 1G, 1B or 1C (short tailed isoforms). Myosin 1E co-localizes with CARMIL, FHOD1, Arp3 and β3-integrin in those early adhesions. But these structures precede stable paxillin-rich adhesions. Myosin 1E movement depends upon actin-binding domains and the presence of an SH3 oligomerization domain. Overexpression of a Myosin 1E deletion mutant without the extreme C-terminal interacting (SH3) domain (Myosin 1EΔSH3) increases edge fluctuations and decreases stable adhesion lifetimes. In contrast, overexpression of Myosin 1E full tail domain (TH1+TH2+TH3/SH3) decreases edge fluctuation. In Myosin 1E knockdown cells, and more prominently in cells treated with Myosin 1 inhibitor, cell–matrix adhesions are also short-lived and fail to mature. We suggest that, by moving to actin polymerization sites and early adhesion sites in active lamellipodia, Myosin 1E might play important roles in transporting not only important polymerizing proteins but also proteins involved in adhesion stabilization. PMID:24337113

  11. Class I Myosins Have Overlapping and Specialized Functions in Left-Right Asymmetric Development in Drosophila

    PubMed Central

    Okumura, Takashi; Sasamura, Takeshi; Inatomi, Momoko; Hozumi, Shunya; Nakamura, Mitsutoshi; Hatori, Ryo; Taniguchi, Kiichiro; Nakazawa, Naotaka; Suzuki, Emiko; Maeda, Reo; Yamakawa, Tomoko; Matsuno, Kenji

    2015-01-01

    The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left–right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes. PMID:25659376

  12. 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

  13. Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

    PubMed Central

    Guilford, W H; Dupuis, D E; Kennedy, G; Wu, J; Patlak, J B; Warshaw, D M

    1997-01-01

    Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin. Images FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 PMID:9138552

  14. Colorimetric Assays of Na,K-ATPase.

    PubMed

    Sweadner, Kathleen J

    2016-01-01

    The Na,K-ATPase is a plasma membrane enzyme that catalyzes active ion transport by the hydrolysis of ATP. Its activity in vivo is determined by many factors, particularly the concentration of intracellular sodium ions. It is the target of the cardiac glycoside class of drugs and of endogenous regulators. Its assay is often an endpoint in the investigation of physiological processes, and it is a promising drug target. As described in this unit, its enzymatic activity can be determined in extracts from tissues by test tube assay using a spectrophotometer or (32)P-ATP. The protocols in this chapter measure inorganic phosphate as the end product of hydrolysis of ATP. PMID:26695025

  15. Specific Evolution of F1-Like ATPases in Mycoplasmas

    PubMed Central

    Dautant, Alain; Bouyssou, Guillaume; Labroussaa, Fabien; Sköllermo, Anna; Persson, Anja; Blanchard, Alain; Sirand-Pugnet, Pascal

    2012-01-01

    F1F0 ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, β, γ and ε subunits of F1 ATPases and could form an F1-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F1-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F1-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F1-like structure is associated with a hypothetical X0 sector located in the membrane of mycoplasma cells. PMID:22685606

  16. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    PubMed

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  17. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  18. C-peptide, Na+,K(+)-ATPase, and diabetes.

    PubMed

    Vague, P; Coste, T C; Jannot, M F; Raccah, D; Tsimaratos, M

    2004-01-01

    Na+,K(+)-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na+,K(+)-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na+,K(+)-ATPase activity was strongly related to blood C-peptide levels in non-insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene. A polymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na+,K(+)-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na+,K(+)-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na+,K(+)-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na+,K(+)-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na+,K(+)-ATPase activity. This impairment in Na+,K(+)-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetes-induced decrease in Na+,K(+)-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na+,K(+)-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na+,K(+)-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na+,K(+)-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications. PMID:15198370

  19. Proton pump activity and Mg-ATPase activity in rat kidney cortex brushborder membranes: effect of 'proton ATPase' inhibitors.

    PubMed

    Kinne-Saffran, E; Kinne, R

    1986-01-01

    In order to further characterize the ATP driven proton pump present in the luminal membrane of the renal proximal tubule, brushborder membranes were isolated from rat kidney cortex and the effect of various proton ATPase inhibitors on intravesicular ATP hydrolysis in sealed brushborder membrane vesicles and on Mg-ATPase activity in permeabilized brushborder membranes was investigated. The protonophor induced intravesicular ATP hydrolysis (ATP driven proton pump) was inhibited by DCCD and filipin but not by diethylstilbestrol and duramycin. All four compounds decreased Mg-ATPase activity, the two former inhibited the ATPase activity with a lower potency than the proton pump. NEM--up to 10 mM--and orthovanadate did not affect intravesicular ATP hydrolysis nor Mg-ATPase activity. From the relative sensitivity of the proton pump and the Mg-ATPase activity to the inhibitors it is concluded that about 35% of the Mg-ATPase activity found in the brushborder membrane can be attributed to the ATP-driven proton pump. Furthermore, the results obtained with NEM and duramycin suggest that the brushborder membrane proton pump has different properties than the proton pump in clathrin-coated vesicles or endosomes. The results presented above raise the possibility that the brushborder membrane proton pump is predominantly involved in acid secretion by the proximal convoluted tubule whereas the proton pump in clathrin-coated vesicles may be predominantly involved in the endocytosis of larger peptides and proteins. PMID:2881248

  20. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy

    PubMed Central

    Hayek, Summer R.; Lee, Samuel A.; Parra, Karlett J.

    2014-01-01

    Vacuolar proton-translocating ATPase (V-ATPase) is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens. PMID:24478704

  1. Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

    PubMed

    Zhou, Aimin; Bu, Yuanyuan; Takano, Tetsuo; Zhang, Xinxin; Liu, Shenkui

    2016-01-01

    In plant cells, the vacuolar-type H(+) -ATPases (V-ATPase) are localized in the tonoplast, Golgi, trans-Golgi network and endosome. However, little is known about how V-ATPase influences plant growth, particularly with regard to the V-ATPase c subunit (VHA-c). Here, we characterized the function of a VHA-c gene from Puccinellia tenuiflora (PutVHA-c) in plant growth. Compared to the wild-type, transgenic plants overexpressing PutVHA-c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V-ATPase activity. Consistently, the Arabidopsis atvha-c5 mutant shows reduced V-ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V-ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA-c-GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling-1 (AtRGS1). These findings suggest that the decrease in V-ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA-c plays an important role in plant growth by influencing V-ATPase-dependent endosomal trafficking. PMID:25917395

  2. Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

    PubMed

    Spudich, James A; Aksel, Tural; Bartholomew, Sadie R; Nag, Suman; Kawana, Masataka; Yu, Elizabeth Choe; Sarkar, Saswata S; Sung, Jongmin; Sommese, Ruth F; Sutton, Shirley; Cho, Carol; Adhikari, Arjun S; Taylor, Rebecca; Liu, Chao; Trivedi, Darshan; Ruppel, Kathleen M

    2016-01-01

    Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. PMID:26792326

  3. Using fluorescent myosin to directly visualize cooperative activation of thin filaments.

    PubMed

    Desai, Rama; Geeves, Michael A; Kad, Neil M

    2015-01-23

    Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin's access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion. PMID:25429108

  4. The role of myosin in vesicle transport during bovine chromaffin cell secretion.

    PubMed Central

    Neco, Patricia; Gil, Anabel; Del Mar Francés, María; Viniegra, Salvador; Gutiérrez, Luis M

    2002-01-01

    Bovine adrenomedullary cells in culture have been used to study the role of myosin in vesicle transport during exocytosis. Amperometric determination of calcium-dependent catecholamine release from individual digitonin-permeabilized cells treated with 3 microM wortmannin or 20 mM 2,3-butanedione monoxime (BDM) and stimulated by continuous as well as repetitive calcium pulses showed alteration of slow phases of secretion when compared with control untreated cells. The specificity of these drugs for myosin inhibition was further supported by the use of peptide-18, a potent peptide affecting myosin light-chain kinase activity. These results were supported also by studying the impact of these myosin inhibitors on chromaffin granule mobility using direct visualization by dynamic confocal microscopy. Wortmannin and BDM affect drastically vesicle transport throughout the cell cytoplasm, including the region beneath the plasma membrane. Immunocytochemical studies demonstrate the presence of myosin types II and V in the cell periphery. The capability of antibodies to myosin II in abrogating the secretory response from populations of digitonin-permeabilized cells compared with the modest effect caused by anti-myosin V suggests that myosin II plays a fundamental role in the active transport of vesicles occurring in the sub-plasmalemmal area during chromaffin cell secretory activity. PMID:12225290

  5. 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. PMID:26754648

  6. Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis.

    PubMed Central

    Halsell, S R; Kiehart, D P

    1998-01-01

    Drosophila is an ideal metazoan model system for analyzing the role of nonmuscle myosin-II (henceforth, myosin) during development. In Drosophila, myosin function is required for cytokinesis and morphogenesis driven by cell migration and/or cell shape changes during oogenesis, embryogenesis, larval development and pupal metamorphosis. The mechanisms that regulate myosin function and the supramolecular structures into which myosin incorporates have not been systematically characterized. The genetic screens described here identify genomic regions that uncover loci that facilitate myosin function. The nonmuscle myosin heavy chain is encoded by a single locus, zipper. Contiguous chromosomal deficiencies that represent approximately 70% of the euchromatic genome were screened for genetic interactions with two recessive lethal alleles of zipper in a second-site noncomplementation assay for the malformed phenotype. Malformation in the adult leg reflects aberrations in cell shape changes driven by myosin-based contraction during leg morphogenesis. Of the 158 deficiencies tested, 47 behaved as second-site noncomplementors of zipper. Two of the deficiencies are strong interactors, 17 are intermediate and 28 are weak. Finer genetic mapping reveals that mutations in cytoplasmic tropomyosin and viking (collagen IV) behave as second-site noncomplementors of zipper during leg morphogenesis and that zipper function requires a previously uncharacterized locus, E3.10/J3.8, for leg morphogenesis and viability. PMID:9560399

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

    PubMed Central

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

    2000-01-01

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

  8. Mechanical coupling between myosin molecules causes differences between ensemble and single-molecule measurements.

    PubMed

    Walcott, Sam; Warshaw, David M; Debold, Edward P

    2012-08-01

    In contracting muscle, individual myosin molecules function as part of a large ensemble, hydrolyzing ATP to power the relative sliding of actin filaments. The technological advances that have enabled direct observation and manipulation of single molecules, including recent experiments that have explored myosin's force-dependent properties, provide detailed insight into the kinetics of myosin's mechanochemical interaction with actin. However, it has been difficult to reconcile these single-molecule observations with the behavior of myosin in an ensemble. Here, using a combination of simulations and theory, we show that the kinetic mechanism derived from single-molecule experiments describes ensemble behavior; but the connection between single molecule and ensemble is complex. In particular, even in the absence of external force, internal forces generated between myosin molecules in a large ensemble accelerate ADP release and increase how far actin moves during a single myosin attachment. These myosin-induced changes in strong binding lifetime and attachment distance cause measurable properties, such as actin speed in the motility assay, to vary depending on the number of myosin molecules interacting with an actin filament. This ensemble-size effect challenges the simple detachment limited model of motility, because even when motility speed is limited by ADP release, increasing attachment rate can increase motility speed. PMID:22947866

  9. Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis.

    PubMed

    Kerridge, Stephen; Munjal, Akankshi; Philippe, Jean-Marc; Jha, Ankita; de Las Bayonas, Alain Garcia; Saurin, Andrew J; Lecuit, Thomas

    2016-03-01

    Polarized cell shape changes during tissue morphogenesis arise by controlling the subcellular distribution of myosin II. For instance, during Drosophila melanogaster gastrulation, apical constriction and cell intercalation are mediated by medial-apical myosin II pulses that power deformations, and polarized accumulation of myosin II that stabilizes these deformations. It remains unclear how tissue-specific factors control different patterns of myosin II activation and the ratchet-like myosin II dynamics. Here we report the function of a common pathway comprising the heterotrimeric G proteins Gα12/13, Gβ13F and Gγ1 in activating and polarizing myosin II during Drosophila gastrulation. Gα12/13 and the Gβ13F/γ1 complex constitute distinct signalling modules, which regulate myosin II dynamics medial-apically and/or junctionally in a tissue-dependent manner. We identify a ubiquitously expressed GPCR called Smog required for cell intercalation and apical constriction. Smog functions with other GPCRs to quantitatively control G proteins, resulting in stepwise activation of myosin II and irreversible cell shape changes. We propose that GPCR and G proteins constitute a general pathway for controlling actomyosin contractility in epithelia and that the activity of this pathway is polarized by tissue-specific regulators. PMID:26780298

  10. Protein Phosphatase 1 β Paralogs Encode the Zebrafish Myosin Phosphatase Catalytic Subunit

    PubMed Central

    Jayashankar, Vaishali; Nguyen, Michael J.; Carr, Brandon W.; Zheng, Dale C.; Rosales, Joseph B.; Rosales, Joshua B.; Weiser, Douglas C.

    2013-01-01

    Background The myosin phosphatase is a highly conserved regulator of actomyosin contractility. Zebrafish has emerged as an ideal model system to study the in vivo role of myosin phosphatase in controlling cell contractility, cell movement and epithelial biology. Most work in zebrafish has focused on the regulatory subunit of the myosin phosphatase called Mypt1. In this work, we examined the critical role of Protein Phosphatase 1, PP1, the catalytic subunit of the myosin phosphatase. Methodology/Principal Findings We observed that in zebrafish two paralogous genes encoding PP1β, called ppp1cba and ppp1cbb, are both broadly expressed during early development. Furthermore, we found that both gene products interact with Mypt1 and assemble an active myosin phosphatase complex. In addition, expression of this complex results in dephosphorylation of the myosin regulatory light chain and large scale rearrangements of the actin cytoskeleton. Morpholino knock-down of ppp1cba and ppp1cbb results in severe defects in morphogenetic cell movements during gastrulation through loss of myosin phosphatase function. Conclusions/Significance Our work demonstrates that zebrafish have two genes encoding PP1β, both of which can interact with Mypt1 and assemble an active myosin phosphatase. In addition, both genes are required for convergence and extension during gastrulation and correct dosage of the protein products is required. PMID:24040418

  11. Myosin XI Is Essential for Tip Growth in Physcomitrella patens[W

    PubMed Central

    Vidali, Luis; Burkart, Graham M.; Augustine, Robert C.; Kerdavid, Erin; Tüzel, Erkan; Bezanilla, Magdalena

    2010-01-01

    Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells. PMID:20525854

  12. Flexibility within the Heads of Muscle Myosin-2 Molecules☆

    PubMed Central

    Billington, Neil; Revill, Derek J.; Burgess, Stan A.; Chantler, Peter D.; Knight, Peter J.

    2014-01-01

    We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN·nm/rad2 is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor–lever junction. PMID:24333017

  13. In Vivo Orientation of Single Myosin Lever Arms in Zebrafish Skeletal Muscle

    PubMed Central

    Sun, Xiaojing; Ekker, Stephen C.; Shelden, Eric A.; Takubo, Naoko; Wang, Yihua; Burghardt, Thomas P.

    2014-01-01

    Cardiac and skeletal myosin assembled in the muscle lattice power contraction by transducing ATP free energy into the mechanical work of moving actin. Myosin catalytic/lever-arm domains comprise the transduction/mechanical coupling machinery that move actin by lever-arm rotation. In vivo, myosin is crowded and constrained by the fiber lattice as side chains are mutated and otherwise modified under normal, diseased, or aging conditions that collectively define the native myosin environment. Single-myosin detection uniquely defines bottom-up characterization of myosin functionality. The marriage of in vivo and single-myosin detection to study zebrafish embryo models of human muscle disease is a multiscaled technology that allows one-to-one registration of a selected myosin molecular alteration with muscle filament-sarcomere-cell-fiber-tissue-organ- and organism level phenotypes. In vivo single-myosin lever-arm orientation was observed at superresolution using a photoactivatable-green-fluorescent-protein (PAGFP)-tagged myosin light chain expressed in zebrafish skeletal muscle. By simultaneous observation of multiphoton excitation fluorescence emission and second harmonic generation from myosin, we demonstrated tag specificity for the lever arm. Single-molecule detection used highly inclined parallel beam illumination and was verified by quantized photoactivation and photobleaching. Single-molecule emission patterns from relaxed muscle in vivo provided extensive superresolved dipole orientation constraints that were modeled using docking scenarios generated for the myosin (S1) and GFP crystal structures. The dipole orientation data provided sufficient constraints to estimate S1/GFP coordination. The S1/GFP coordination in vivo is rigid and the lever-arm orientation distribution is well-ordered in relaxed muscle. For comparison, single myosins in relaxed permeabilized porcine papillary muscle fibers indicated slightly differently oriented lever arms and rigid S1/GFP coordination. Lever arms in both muscles indicated one preferred spherical polar orientation and widely distributed azimuthal orientations relative to the fiber symmetry axis. Cardiac myosin is more radially displaced from the fiber axis. Probe rigidity implies the PAGFP tag reliably indicates cross-bridge orientation in situ and in vivo. PMID:25229148

  14. Conformation of the myosin motor during force generation in skeletal muscle.

    PubMed

    Irving, M; Piazzesi, G; Lucii, L; Sun, Y B; Harford, J J; Dobbie, I M; Ferenczi, M A; Reconditi, M; Lombardi, V

    2000-06-01

    Myosin motors drive muscle contraction, cytokinesis and cell locomotion, and members of the myosin superfamily have been implicated in an increasingly diverse range of cell functions. Myosin can displace a bound actin filament several nanometers in a single interaction. Crystallographic studies suggest that this 'working stroke' involves bending of the myosin head between its light chain and catalytic domains. Here we used X-ray fiber diffraction to test the crystallographic model and measure the interdomain bending during force generation in an intact single muscle fiber. The observed bending has two components: an elastic distortion and an active rotation that generates force. The average bend of the force-generating myosin heads in a muscle fiber is intermediate between those in crystal structures with different bound nucleotides, and the C-terminus of the head is displaced by 7 nm along the actin filament axis compared with the in vitro conformation seen in the absence of nucleotide. PMID:10881196

  15. Catch-bond behaviour facilitates membrane tubulation by non-processive myosin 1b.

    PubMed

    Yamada, Ayako; Mamane, Alexandre; Lee-Tin-Wah, Jonathan; Di Cicco, Aurélie; Prévost, Coline; Lévy, Daniel; Joanny, Jean-François; Coudrier, Evelyne; Bassereau, Patricia

    2014-01-01

    Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes. PMID:24709651

  16. How are the cellular functions of myosin VI regulated within the cell?

    PubMed Central

    Buss, Folma; Kendrick-Jones, John

    2008-01-01

    This review, dedicated to the memory of Professor Setsuro Ebashi, focuses on our current work investigating the cellular functions and regulation of the unique unconventional motor, myosin VI. This myosin, unlike all the other myosins so far studied, moves towards the minus end of actin filaments and has been implicated in a wide range of cellular processes such as endocytosis, exocytosis, cell migration, cell division and cytokinesis. Myosin VI’s involvement in these cellular pathways is mediated by its interaction with specific adaptor proteins and is regulated by multiple regulatory signals and modifications such as calcium ions, PtdIns(4,5)P2 (PIP2) and phosphorylation. Understanding the functions of myosin VI within the cell and how it is regulated is now of utmost importance given the recent observations that it is associated with a number of human disorders such as deafness and cancers. PMID:18068125

  17. 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.

  18. Catch-bond behaviour facilitates membrane tubulation by non-processive myosin 1b

    NASA Astrophysics Data System (ADS)

    Yamada, Ayako; Mamane, Alexandre; Lee-Tin-Wah, Jonathan; di Cicco, Aurélie; Prévost, Coline; Lévy, Daniel; Joanny, Jean-François; Coudrier, Evelyne; Bassereau, Patricia

    2014-04-01

    Myosin 1b is a single-headed membrane-associated motor that binds to actin filaments with a catch-bond behaviour in response to load. In vivo, myosin 1b is required to form membrane tubules at both endosomes and the trans-Golgi network. To establish the link between these two fundamental properties, here we investigate the capacity of myosin 1b to extract membrane tubes along bundled actin filaments in a minimal reconstituted system. We show that single-headed non-processive myosin 1b can extract membrane tubes at a biologically relevant low density. In contrast to kinesins we do not observe motor accumulation at the tip, suggesting that the underlying mechanism for tube formation is different. In our theoretical model, myosin 1b catch-bond properties facilitate tube extraction under conditions of increasing membrane tension by reducing the density of myo1b required to pull tubes.

  19. Biochemical and bioinformatic analysis of the MYO19 motor domain

    PubMed Central

    Adikes, Rebecca C.; Unrath, William C.; Yengo, Christopher M.; Quintero, Omar A.

    2014-01-01

    Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-cosedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment. PMID:23568824

  20. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening

    PubMed Central

    Elbatsh, Ahmed M.O.; Haarhuis, Judith H.I.; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H.; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, René H.; Nasmyth, Kim; Rowland, Benjamin D.

    2016-01-01

    Summary Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin’s Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin’s highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin’s two ATPase sites. PMID:26895426

  1. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening.

    PubMed

    Elbatsh, Ahmed M O; Haarhuis, Judith H I; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, Ren H; Nasmyth, Kim; Rowland, Benjamin D

    2016-02-18

    Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites. PMID:26895426

  2. Multiple functions of Na,K-ATPase in epithelial cells.

    PubMed

    Rajasekaran, Sigrid A; Barwe, Sonali P; Rajasekaran, Ayyappan K

    2005-09-01

    The Na,K-adenosine triphosphatase (ATPase), or sodium pump, has been well studied for its role in the regulation of ion homeostasis in mammalian cells. Recent studies suggest that Na,K-ATPase might have multiple functions such as a role in the regulation of tight junction structure and function, induction of polarity, regulation of actin dynamics, control of cell movement, and cell signaling. These functions appear to be modulated by Na,K-ATPase enzyme activity as well as protein-protein interactions of the alpha and beta subunits. In this review we attempt to differentiate functions associated with enzyme activity and subunit interactions. In addition, the consequence of impaired Na,K-ATPase function or reduced subunit expression levels in kidney diseases such as cancer, tubulointerstitial fibrosis, and ischemic nephropathy are discussed. PMID:16139688

  3. Regulation of Torsin ATPases by LAP1 and LULL1.

    PubMed

    Zhao, Chenguang; Brown, Rebecca S H; Chase, Anna R; Eisele, Markus R; Schlieker, Christian

    2013-04-23

    TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder. PMID:23569223

  4. Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex

    PubMed Central

    Le, Kang; Li, Chun-Chun; Ye, Guan; Moss, Joel; Vaughan, Martha

    2013-01-01

    Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells. PMID:23918382

  5. Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin.

    PubMed

    Abel, L C; Kalil, J; Cunha Neto, E

    1997-11-01

    Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas' disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas' cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI = 25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas' disease cardiomyopathy. PMID:9532238

  6. A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

    PubMed Central

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

    2010-01-01

    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. PMID:20084115

  7. 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.

  8. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    SciTech Connect

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera; Hofmann, Wilma A.

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

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

    PubMed Central

    Betapudi, Venkaiah

    2014-01-01

    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. PMID:25072053

  10. Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length

    PubMed Central

    Suggs, Jennifer A.; Cammarato, Anthony; Kronert, William A.; Nikkhoy, Massoud; Dambacher, Corey M.; Megighian, Aram; Bernstein, Sanford I.

    2007-01-01

    Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin “hinge” region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial “melting” of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was ~5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed. PMID:17316684

  11. Neutral Phospholipids Stimulate Na,K-ATPase Activity

    PubMed Central

    Haviv, Haim; Habeck, Michael; Kanai, Ryuta; Toyoshima, Chikashi; Karlish, Steven J. D.

    2013-01-01

    Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8–10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8–10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid. PMID:23430748

  12. Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

    PubMed

    Tanner, Bertrand C W; Breithaupt, Jason J; Awinda, Peter O

    2015-12-15

    Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 μm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 μm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 μm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 μm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart. PMID:26475586

  13. Evidence for functional differences between two flagellar dynein ATPases.

    PubMed

    Penningroth, S M; Peterson, D D

    1986-01-01

    Energy coupling in flagellar motility was investigated using demembranated, reactivated sea urchin spermatozoa (Arbacia punctulata). The ATP-dependence of ATPase activity was investigated for ATP concentrations ranging from 4 microM to 600 microM ATP. Using Eadie-Scatchard plot analysis, we identified two axonemal dynein ATPase activities. Their apparent Michaelis constants were calculated to be equal to 4 microM and 161 microM ATP, and they were referred to, respectively, as the high-affinity dynein ATPase (HADA) and the low-affinity dynein ATPase (LADA). Investigation of movement-coupled ATPase activity (difference between the ATPase activities of reactivated and broken, immotile spermatozoa) indicated that HADA and LADA were both 65% movement-coupled. The apparent Michaelis constants of movement-coupled HADA and LADA, 12 microM and 271 microM ATP, respectively, were two- to four-fold greater than the apparent Michaelis constants of movement-uncoupled HADA and LADA. The apparent Michaelis constants for force generation and beat frequency of reactivated spermatozoa were determined to be 24 microM and 290 microM ATP, respectively. These results raise the possibility that flagellar force generation is controlled primarily by movement-coupled HADA, and that flagellar beat frequency is controlled primarily by movement-coupled LADA. Thus, mechanochemical activity in flagellar motility may be divided between two enzymatically and functionally distinct classes of flagellar dyneins. PMID:2948677

  14. Unconventional processive mechanics of non-muscle myosin IIB.

    PubMed

    Norstrom, Melanie F; Smithback, Philip A; Rock, Ronald S

    2010-08-20

    Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of -5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension. PMID:20511646

  15. Par-4: A New Activator of Myosin Phosphatase

    PubMed Central

    Vetterkind, Susanne; Lee, Eunhee; Sundberg, Eric; Poythress, Ransom H.; Tao, Terence C.; Preuss, Ute

    2010-01-01

    Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility. Here, we test the hypothesis that Par-4 regulates MP activity directly. We show, by proximity ligation assays, surface plasmon resonance and coimmunoprecipitation, that Par-4 interacts with the targeting subunit of MP, MYPT1. Binding is mediated by the leucine zippers of MYPT1 and Par-4 and reduced by Par-4 phosphorylation. Overexpression of Par-4 leads to increased phosphatase activity of immunoprecipitated MP, whereas small interfering RNA knockdown of endogenous Par-4 significantly decreases MP activity and increases MYPT1 phosphorylation. LC20 phosphorylation assays demonstrate that overexpression of Par-4 reduces LC20 phosphorylation. In contrast, a phosphorylation site mutant, but not wild-type Par-4, interferes with zipper-interacting protein kinase (ZIPK)-mediated MP inhibition. We conclude from our results Par-4 operates through a “padlock” model in which binding of Par-4 to MYPT1 activates MP by blocking access to the inhibitory phosphorylation sites, and inhibitory phosphorylation of MYPT1 by ZIPK requires “unlocking” of Par-4 by phosphorylation and displacement of Par-4 from the MP complex. PMID:20130087

  16. 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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  18. Calcium can mobilize and activate myosin-VI.

    PubMed

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

    2016-03-01

    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

  19. Ouabain binding sites and (Na/sup +/,K/sup +/)-ATPase activity in rat cardiac hypertrophy: expression of the neonatal forms

    SciTech Connect

    Not Available

    1986-01-05

    The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involved several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of (/sup 3/H)ouabain and ouabain-induced inhibition of (Na/sup +/,K/sup +/)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (K/sub d/) of 1.8-3.2 x 10/sup -8/ M and 1-8 x 10/sup -6/ M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na/sup +/,K/sup +/)-ATPase (IC/sub 50/ = 1-3 x 10/sup -8/ M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms, the data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor.

  20. Cadmium inhibits motility, activities of plasma membrane Ca(2+) -ATPase and axonemal dynein-ATPase of human spermatozoa.

    PubMed

    Da Costa, R; Botana, D; Piñero, S; Proverbio, F; Marín, R

    2016-05-01

    Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+) -ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+) -ATPase reached maximal inhibition at 50 nm free Cd(2+) , with a K50% inhibition of 18.3 nm free Cd(2+) . The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+) . Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+) . PMID:26259968

  1. Nanometric features of myosin filaments extracted from a single muscle fiber to uncover the mechanisms underlying organized motility.

    PubMed

    Li, Meishan; Deguchi, Takahiro; Näreoja, Tuomas; Jena, Bhanu P; Hänninen, Pekka; Larsson, Lars

    2015-10-01

    The single muscle fiber in vitro motility assay (SF-IVMA) is characterized by organized linear motility of actin filaments, i.e., actin filaments motility showing a parallel or anti-parallel direction with similar speed independent of direction in the central part of the flow-cell where density of myosin is high. In contrast, the low myosin density region in the flow-cell exhibits random filament movements, but the mechanisms underlying the organized motility remain unknown. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging techniques have been combined to investigate the morphological features of myosin extracted from single muscle fiber segments in the flow cell. Nanometric scale imaging of myosin filaments in the SF-IVMA showed intact spatial distances between myosin heads being essential for myosin filament function. However, angular spectrum analyses of myosin filaments in the high myosin density region showed organized myosin filament orientation only in small areas, while unorganized filament orientation were dominantly presented when larger areas were analyzed. Thus, parallel myosin filament organization is a less likely mechanism underlying the organized motility of actin filaments and the high myosin density per se is therefore forwarded as the primary "driver" that promotes organized linear motility. PMID:26116379

  2. 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

  3. Evolutionary appearance of the plasma membrane H (+) -ATPase containing a penultimate threonine in the bryophyte.

    PubMed

    Okumura, Masaki; Takahashi, Koji; Inoue, Shin-Ichiro; Kinoshita, Toshinori

    2012-08-01

    The plasma membrane H (+) -ATPase provides the driving force for solute transport via an electrochemical gradient of H (+) across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H (+) -ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H (+) -ATPase (pT H (+) -ATPase) and non-pT H (+) -ATPase as in the green algae, and that pT H (+) -ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H (+) -ATPase genes, designated PpHA (Physcomitrella patens H (+) -ATPase). Six isoforms are the pT H (+) -ATPase; a remaining isoform is non-pT H (+) -ATPase. An apparent 95-kD protein was recognized by anti-H (+) -ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H (+) -ATPase. Furthermore, we could not detect the pT H (+) -ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H (+) -ATPase most likely appeared for the first time in bryophyte. PMID:22836495

  4. Processive steps in the reverse direction require uncoupling of the lead head lever arm of myosin VI.

    PubMed

    Ménétrey, Julie; Isabet, Tatiana; Ropars, Virginie; Mukherjea, Monalisa; Pylypenko, Olena; Liu, Xiaoyan; Perez, Javier; Vachette, Patrice; Sweeney, H Lee; Houdusse, Anne M

    2012-10-12

    Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model. PMID:22940248

  5. Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

    PubMed

    Russell, V E; Klein, U; Reuveni, M; Spaeth, D D; Wolfersberger, M G; Harvey, W R

    1992-05-01

    In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources. PMID:1534830

  6. Pharmacological activation of myosin II paralogs to correct cell mechanics defects.

    PubMed

    Surcel, Alexandra; Ng, Win Pin; West-Foyle, Hoku; Zhu, Qingfeng; Ren, Yixin; Avery, Lindsay B; Krenc, Agata K; Meyers, David J; Rock, Ronald S; Anders, Robert A; Freel Meyers, Caren L; Robinson, Douglas N

    2015-02-01

    Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. PMID:25605895

  7. Involvement of myosin light-chain kinase in endothelial cell retraction

    SciTech Connect

    Wysolmerski, R.B.; Lagunoff, D. )

    1990-01-01

    Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.

  8. Pharmacological activation of myosin II paralogs to correct cell mechanics defects

    PubMed Central

    Surcel, Alexandra; Ng, Win Pin; West-Foyle, Hoku; Zhu, Qingfeng; Ren, Yixin; Avery, Lindsay B.; Krenc, Agata K.; Meyers, David J.; Rock, Ronald S.; Anders, Robert A.; Freel Meyers, Caren L.; Robinson, Douglas N.

    2015-01-01

    Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior. PMID:25605895

  9. 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

  10. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division

    PubMed Central

    Uhart, Perrine; Tassan, Jean-Pierre; Michaux, Grégoire

    2015-01-01

    During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis. PMID:26416962

  11. 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.

  12. High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label.

    PubMed

    Binder, Benjamin P; Cornea, Sinziana; Thompson, Andrew R; Moen, Rebecca J; Thomas, David D

    2015-06-30

    Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin's catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures. PMID:26056276

  13. ALPK1 phosphorylates myosin IIA modulating TNF-α trafficking in gout flares

    PubMed Central

    Lee, Chi-Pin; Chiang, Shang-Lun; Ko, Albert Min-Shan; Liu, Yu-Fan; Ma, Che; Lu, Chi-Yu; Huang, Chung-Ming; Chang, Jan-Gowth; Kuo, Tzer-Min; Chen, Chia-Lin; Tsai, Eing-Mei; Ko, Ying-Chin

    2016-01-01

    Gout is characterized by the monosodium urate monohydrate (MSU)-induced arthritis. Alpha kinase-1 (ALPK1) has shown to be associated with MSU-induced inflammation and gout. Here, we used bioinformatics, proteomics, cell models, and twenty in vitro human assays to clarify some of its role in the inflammatory response to MSU. We found myosin IIA to be a frequent interacting protein partner of ALPK1, binding to its N-terminal and forming a protein complex with calmodulin and F-actin, and that MSU-induced ALPK1 phosphorylated the myosin IIA. A knockdown of endogenous ALPK1 or myosin IIA significantly reduced the MSU-induced secretion of tumour necrosis factor (TNF)-α. Furthermore, all gouty patients expressed higher basal protein levels of ALPK1, myosin IIA, and plasma TNF-α, however those medicated with colchicine has shown reduced myosin IIA and TNF-α but not ALPK1. The findings suggest ALPK1 is a kinase that participates in the regulation of Golgi-derived TNF-α trafficking through myosin IIA phosphorylation in the inflammation of gout. This novel pathway could be blocked at the level of myosin by colchicine in gout treatment. PMID:27169898

  14. 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

  15. 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.

  16. Overexpression of myosin IB in living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis.

    PubMed

    Marion, Sabrina; Wilhelm, Claire; Voigt, Heike; Bacri, Jean-Claude; Guillén, Nancy

    2004-07-01

    The human parasite Entamoeba histolytica is an ancient protozoan that expresses only one unconventional myosin, which has homology with myosin IB from other amoebae. Myosin IB is involved in phagocytosis of human cells by E. histolytica. In this work, we developed a microrheological technique, analysing magnetic phagosomes, which allowed us to probe the density of the F-actin network in living cells. Using this technique, we showed that overexpression of myosin IB led to an increase in cytoplasm viscosity, which correlated with a delay in initiating human cell phagocytosis. To investigate which myosin IB domains sustain cell viscosity changes, we overexpressed truncated forms of the protein. Our results demonstrate that both actin-binding sites that are present in the heavy chain but not the SH3 domain are required to modulate the density of the actin network. These data suggested that, as well as the motor activity, myosin IB in E. histolytica plays a structural role on the actin network owing to its ability to cross-link filaments. The gelation state of cell cytoplasm and the dynamics of cortical F-actin during phagocytosis seem to be modulated by the myosin IB structuring cytoskeleton activity. PMID:15226399

  17. The power stroke of myosin VI and the basis of reverse directionality.

    PubMed

    Bryant, Zev; Altman, David; Spudich, James A

    2007-01-16

    Myosin VI supports movement toward the (-) end of actin filaments, despite sharing extensive sequence and structural homology with (+)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120 degrees redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Ménétrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005) Nature 435:779-785]. However, this redirection alone is insufficient to account for the large (-)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (+)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (+)-end-directed motion, whereas longer constructs move toward the (-) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (-) end. We suggest that the lever arm rotates approximately 180 degrees between pre- and poststroke conformations. PMID:17182734

  18. 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.

  19. ALPK1 phosphorylates myosin IIA modulating TNF-α trafficking in gout flares.

    PubMed

    Lee, Chi-Pin; Chiang, Shang-Lun; Ko, Albert Min-Shan; Liu, Yu-Fan; Ma, Che; Lu, Chi-Yu; Huang, Chung-Ming; Chang, Jan-Gowth; Kuo, Tzer-Min; Chen, Chia-Lin; Tsai, Eing-Mei; Ko, Ying-Chin

    2016-01-01

    Gout is characterized by the monosodium urate monohydrate (MSU)-induced arthritis. Alpha kinase-1 (ALPK1) has shown to be associated with MSU-induced inflammation and gout. Here, we used bioinformatics, proteomics, cell models, and twenty in vitro human assays to clarify some of its role in the inflammatory response to MSU. We found myosin IIA to be a frequent interacting protein partner of ALPK1, binding to its N-terminal and forming a protein complex with calmodulin and F-actin, and that MSU-induced ALPK1 phosphorylated the myosin IIA. A knockdown of endogenous ALPK1 or myosin IIA significantly reduced the MSU-induced secretion of tumour necrosis factor (TNF)-α. Furthermore, all gouty patients expressed higher basal protein levels of ALPK1, myosin IIA, and plasma TNF-α, however those medicated with colchicine has shown reduced myosin IIA and TNF-α but not ALPK1. The findings suggest ALPK1 is a kinase that participates in the regulation of Golgi-derived TNF-α trafficking through myosin IIA phosphorylation in the inflammation of gout. This novel pathway could be blocked at the level of myosin by colchicine in gout treatment. PMID:27169898

  20. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.

    PubMed

    Pacquelet, Anne; Uhart, Perrine; Tassan, Jean-Pierre; Michaux, Grégoire

    2015-09-28

    During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis. PMID:26416962

  1. SLOW MYOSIN ATP TURNOVER IN THE SUPER-RELAXED STATE IN TARANTULA MUSCLE

    PubMed Central

    Naber, Nariman; Cooke, Roger

    2011-01-01

    We measured the nucleotide turnover rate of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ~30 minutes. This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in mantADP and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with the very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations. PMID:21763701

  2. Myosin content of individual human muscle fibers isolated by laser capture microdissection.

    PubMed

    Stuart, Charles A; Stone, William L; Howell, Mary E A; Brannon, Marianne F; Hall, H Kenton; Gibson, Andrew L; Stone, Michael H

    2016-03-01

    Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. PMID:26676053

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

    SciTech Connect

    Schewkunow, Vitali; Sharma, Karan P.; Diez, Gerold; Klemm, Anna H.; Sharma, Pal C.; Goldmann, Wolfgang H.

    2008-02-08

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

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

    PubMed Central

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

    2014-01-01

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

  6. The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

    PubMed Central

    Noguchi, Taro Q. P.; Morimatsu, Masatoshi; Iwane, Atsuko H.; Yanagida, Toshio; Uyeda, Taro Q. P.

    2015-01-01

    The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin. PMID:25945499

  7. Myosin filament polymerization and depolymerization in a model of partial length adaptation in airway smooth muscle

    PubMed Central

    Ijpma, Gijs; Cairns, Simeon P.; Sieck, Gary C.

    2011-01-01

    Length adaptation in airway smooth muscle (ASM) is attributed to reorganization of the cytoskeleton, and in particular the contractile elements. However, a constantly changing lung volume with tidal breathing (hence changing ASM length) is likely to restrict full adaptation of ASM for force generation. There is likely to be continuous length adaptation of ASM between states of incomplete or partial length adaption. We propose a new model that assimilates findings on myosin filament polymerization/depolymerization, partial length adaptation, isometric force, and shortening velocity to describe this continuous length adaptation process. In this model, the ASM adapts to an optimal force-generating capacity in a repeating cycle of events. Initially the myosin filament, shortened by prior length changes, associates with two longer actin filaments. The actin filaments are located adjacent to the myosin filaments, such that all myosin heads overlap with actin to permit maximal cross-bridge cycling. Since in this model the actin filaments are usually longer than myosin filaments, the excess length of the actin filament is located randomly with respect to the myosin filament. Once activated, the myosin filament elongates by polymerization along the actin filaments, with the growth limited by the overlap of the actin filaments. During relaxation, the myosin filaments dissociate from the actin filaments, and then the cycle repeats. This process causes a gradual adaptation of force and instantaneous adaptation of shortening velocity. Good agreement is found between model simulations and the experimental data depicting the relationship between force development, myosin filament density, or shortening velocity and length. PMID:21659490

  8. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    PubMed

    Monroe, Nicole; Hill, Christopher P

    2016-05-01

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. PMID:26555750

  9. Genetic Variation in Myosin 1H Contributes to Mandibular Prognathism

    PubMed Central

    Tassopoulou-Fishell, Maria; Deeley, Kathleen; Harvey, Erika M.; Sciote, James; Vieira, Alexandre R.

    2013-01-01

    Introduction Several candidate loci have been suggested as influencing mandibular prognathism (1p22.1, 1p22.2, 1p36, 3q26.2, 5p13-p12, 6q25, 11q22.2-q22.3, 12q23, 12q13.13, and 19p13.2). The goal of this study was to replicate these results in a well-characterized homogeneous sample set. Methods Thirty-three single nucleotide polymorphisms spanning all candidate regions were studied in 44 prognathic and 35 Class I subjects from the University of Pittsburgh School of Dental Medicine Dental Registry and DNA Repository. The 44 mandibular prognathism subjects had an average age of 18.4 years, 31 were females and 13 males, and 24 were White, 15 African American, two Hispanic, and three Asian. The 35 Class I subjects had an average age of 17.6 years, 27 were females and 9 males, and 27 were White, six African Americans, one Hispanic, and two Asian. Skeletal mandibular prognathism diagnosis included cephalometric values indicative of Class III such as ANB smaller than two degrees, negative Witts appraisal, and positive A–B plane. Additional mandibular prognathism criteria included negative OJ and visually prognathic (concave) profile as determined by the subject's clinical evaluation. Orthognathic subjects without jaw deformations were used as a comparison group. Mandibular prognathism and orthognathic subjects were matched based on race, sex and age. Genetic markers were tested by polymerase chain reaction using TaqMan chemistry. Chi-square and Fisher exact tests were used to determine overrepresentation of marker allele with alpha of 0.05. Results An association was unveiled between a marker in MYO1H (rs10850110) and the mandibular prognathism phenotype (p=0.03). MYO1H is a Class-I myosin that is in a different protein group than the myosin isoforms of muscle sarcomeres, which are the basis of skeletal muscle fiber typing. Class I myosins are necessary for cell motility, phagocytosis and vesicle transport. Conclusions More strict clinical definitions may increase homogeneity and aid the studies of genetic susceptibility to malocclusions. We provide evidence that MYO1H may contribute to mandibular prognathism. PMID:22196185

  10. Mode of Myosin Transportation in Living Cells Studied by Single Particle Tracking

    NASA Astrophysics Data System (ADS)

    Liang, Zhang-yi; Xu, Ning; Guan, Ying-hua; Zhang, You-yi; Zhao, Xin-sheng

    2007-08-01

    The transport of internalized α1A-adrenergic receptor (α1A-AR) by myosin protein in live cells was studied. The technique of single particle tracking by fluorescence imaging with high temporal and spatial resolution was used. The endosomes of α1A-AR were transported along actin filaments in a step-by-step mode. The average step-size in different time resolutions is consistent with the step-size of myosin assay in vitro. With the simulation of the stepwise traces in different time resolutions, we found that the kinetic process of each step is in coherence with the single myosin assay in vitro.

  11. Myosin motors at neuronal synapses: drivers of membrane transport and actin dynamics.

    PubMed

    Kneussel, Matthias; Wagner, Wolfgang

    2013-04-01

    Myosins are a large family of actin-based cytoskeletal motors that use energy derived from ATP hydrolysis to generate movement and force. Myosins of classes II, V and VI have specific pre- and postsynaptic roles that are required for synapse function. They also facilitate several forms of synaptic plasticity. Interestingly, the myosins of these classes differ markedly in important aspects of their molecular mechanisms of function. Accordingly, their major roles at synapses are diverse and include the regulation of actin cytoskeleton dynamics in dendritic spines and powering of synaptic cargo transport. PMID:23481482

  12. Chaperones of F[subscript 1]-ATPase

    SciTech Connect

    Ludlam, Anthony; Brunzelle, Joseph; Pribyl, Thomas; Xu, Xingjue; Gatti, Domenico L.; Ackerman, Sharon H.

    2009-09-25

    Mitochondrial F{sub 1}-ATPase contains a hexamer of alternating {alpha} and {beta} subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to {beta} and {alpha}. In the absence of Atp11p and Atp12p, the hexamer is not formed, and {alpha} and {beta} precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F{sub 1} assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486--1493) hypothesized that the chaperones themselves look very much like the {alpha} and {beta} subunits, and proposed an exchange of Atp11p for {alpha} and of Atp12p for {beta}; the driving force for the exchange was expected to be a higher affinity of {alpha} and {beta} for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to {beta} and Atp12p is bound to {alpha}, the two F{sub 1} subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to {alpha} and {beta} prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble {alpha} or {beta}, and it is instead the F{sub 1} {gamma} subunit that initiates the release of the chaperones from {alpha} and {beta} and their further assembly into the mature complex.

  13. Myofibrillar ATPase activity in rat heart after chronic propranolol administration.

    PubMed

    Dowell, R T

    1979-11-01

    A previous study has shown that chronic chemical sympathectomy brought about by 6-hydroxydopamine injections results in a dpression in myocardial contractile function which is accompanied by reduced myofibrillar ATPase activity. To determine whether chronic beta-adrenergic receptor blockade elicits similar alterations in cardiac contractile-protein ATPase activity, adult rats were given twice-daily injections of propranolol 7 days/wk for 2 wk. Effective beta-adrenergic receptor blockade was verified by the lack of hemodynamic responsiveness to isoproterenol infusion. Myofibrils were prepared from left ventricular tissue and analyzed for ATPase activity. Myofibrillar ATPase activity was 295 +/- 8 nmol Pi.mg-1.min-1 in controls. Enzyme activity was not significantly different in propranolol-injected rats. The results demonstrate that chronic propranolol administration does not alter the ATPase activity of cardiac myofibrils. Therefore, it seems likely that the altered contractile-protein enzymatic properties resulting from chronic chemical sympathectomy do not occur as the result of a reducted level of cardiac beta-adrenergic receptor stimulation. PMID:158983

  14. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    SciTech Connect

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  15. H/sup +/-translocating ATPases: advances using membrane vesicles

    SciTech Connect

    Sze, H.

    1985-01-01

    In this paper, two primary active transport systems (H/sup +/ -ATPases) in plant cells are examined using membrane vesicles as a simple experimental tool. One electrogenic, H/sup +/ -translocating ATPase is vanadate-sensitive and associated with the plasma membrane. Another electrogenic, H/sup +/ -translocating ATPases is anion-sensitive, and localized on the tonoplast (and perhaps other membranes). According to the working model, the plasma membrane and tonoplast-type H/sup +/ -ATPases are detectable in inside-out plasma membrane and right-side-out tonoplast vesicles. The direction of H/sup +/ pumping into these vesicles would be consistent with the results from intact cells where H/sup +/ are extruded from the cell across the plasma membrane and pumped into the vacuole from the cytoplasm. Understanding the properties of H/sup +/ -pumping ATPases using membrane vesicles has paved the way for studies to identify secondary active transport systems coupled to the proton electrochemical gradient. Redox-driven transport systems can also be studied directly using the isolated vesicles. As transport proteins are identified, the functional activities can be specifically studied after reconstitution of the purified protein(s) into phospholipid membrane vesicles. 154 references.

  16. Thermodynamic efficiency and mechanochemical coupling of F1-ATPase

    PubMed Central

    Toyabe, Shoichi; Watanabe-Nakayama, Takahiro; Okamoto, Tetsuaki; Kudo, Seishi; Muneyuki, Eiro

    2011-01-01

    F1-ATPase is a nanosized biological energy transducer working as part of FoF1-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F1-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F1-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of FoF1-ATP synthase. We found that the maximum work performed by F1-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F1-ATPase. PMID:21997211

  17. Analysis of Methionine Oxidation in Myosin Isoforms in Porcine Skeletal Muscle by LC-MS/MS Analysis

    PubMed Central

    Jeong, Jin-Yeon; Jung, Eun-Young; Jeong, Tae-Chul; Yang, Han-Sul; Kim, Gap-Don

    2016-01-01

    The purpose of this study was to analyze oxidized methionines in the myosin isoforms of porcine longissimus thoracis, psoas major, and semimembranosus muscles by liquid chromatography (LC) and mass spectrometry (MS). A total of 836 queries matched to four myosin isoforms (myosin-1, -2, -4, and -7) were analyzed and each myosin isoform was identified by its unique peptides (7.3-13.3). Forty-four peptides were observed from all three muscles. Seventeen peptides were unique to the myosin isoform and the others were common peptides expressed in two or more myosin isoforms. Five were identified as oxidized peptides with one or two methionine sulfoxides with 16 amu of mass modification. Methionines on residues 215 (215), 438 (438), 853 (851), 856 (854), 1071 (1069), and 1106 (1104) of myosin-1 (myosin-4) were oxidized by the addition of oxygen. Myosin-2 had two oxidized methionines on residues 215 and 438. No queries matched to myosin-7 were observed as oxidized peptides. LC-MS/MS allows analysis of the oxidation of specific amino acids on specific residue sites, as well as in specific proteins in the food system. PMID:27194935

  18. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad.

    PubMed

    Ono, Kanako; Ono, Shoichiro

    2016-04-01

    The myoepithelial sheath in the somatic gonad of the nematodeCaenorhabditis eleganshas nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle-like contractile apparatuses, it has a striated muscle-like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath. PMID:26864628

  19. The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.

    PubMed Central

    Rowe, T; Kendrick-Jones, J

    1993-01-01

    In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified. Images PMID:8223496

  20. Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus.

    PubMed

    Sobczak, Magdalena; Wasik, Anna; Kłopocka, Wanda; Redowicz, Maria Jolanta

    2008-12-01

    Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.). PMID:19088799

  1. Lamellipodial actin mechanically links myosin activity with adhesion-site formation.

    PubMed

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

    2007-02-01

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

  2. 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

  3. An Engineered Minimal WASP-Myosin Fusion Protein Reveals Essential Functions for Endocytosis.

    PubMed

    Lewellyn, Eric B; Pedersen, Ross T A; Hong, Jessica; Lu, Rebecca; Morrison, Huntly M; Drubin, David G

    2015-11-01

    Actin polymerization powers membrane deformation during many processes, including clathrin-mediated endocytosis (CME). During CME in yeast, actin polymerization is triggered and coordinated by a six-protein WASP/Myosin complex that includes WASP, class I myosins (Myo3 and Myo5), WIP (Vrp1), and two other proteins. We show that a single engineered protein can replace this entire complex while still supporting CME. This engineered protein reveals that the WASP/Myosin complex has four essential activities: recruitment to endocytic sites, anchorage to the plasma membrane, Arp2/3 activation, and transient actin filament binding by the motor domain. The requirement for both membrane and F-actin binding reveals that myosin-mediated coupling between actin filaments and the base of endocytic sites is essential for allowing actin polymerization to drive membrane invagination. PMID:26555049

  4. Characterization and localization of dynein and myosins V and VI in the ovaries of queen bees.

    PubMed

    Patricio, Karina; Calábria, Luciana Karen; Peixoto, Pablo Marco; Espindola, Foued Salmen; Da Cruz-Landim, Carminda

    2010-10-01

    The presence of myosin and dynein in the ovaries of both Apis mellifera and Scaptotrigona postica was investigated in extracts and in histological sections. In the ovary extracts, motor proteins, myosins V, VI and dynein were detected by Western blot. In histological sections, they were detected by immunocytochemistry, using a mouse monoclonal antibody against the intermediary chain of dynein and a rabbit polyclonal antibody against the myosin V head domain. The myosin VI tail domain was recognized by a pig polyclonal antibody. The results show that these molecular motors are expressed in the ovaries of both bee species with few differences in location and intensity, in regions where movement of substances is expected during oogenesis. The fact that antibodies against vertebrate proteins recognize proteins of bee species indicates that the specific epitopes are evolutionarily well preserved. PMID:20486900

  5. Simulation model for combined motion of myosin cross-bridges agrees with experimental data.

    PubMed

    Marandos, Peter; Midde, Krishna

    2014-01-01

    The motivation for this work was to derive a theoretical model for the combined motion of a sample of muscle tissue with a small number (approximately 12) of myosin molecules. This was then compared to data collected at the University of North Texas Health Science center. A theoretical model of the motion of the myosin cross-bridges has been derived. The solution is a combination of solutions from the classical harmonic oscillator, Brownian motion, and Maxwell-Boltzmann statistics. The model illustrates the myosin behavior as a function of the number of myosin molecules, the temperature of the sample, and the spring constant. The results show that there is good agreement between the theoretical model and experimental data. PMID:24896359

  6. Hold on tightly, let go lightly: myosin functions at adherens junctions

    PubMed Central

    Sandquist, Joshua C.; Bement, William M.

    2016-01-01

    Adherens junctions, the sites of cadherin-dependent cell–cell adhesion, are also important for dynamic tension sensing, force transduction and signalling. Different myosin motors contribute to adherens junction assembly and versatility in distinct ways. PMID:20596044

  7. Assaying P-Type ATPases Reconstituted in Liposomes.

    PubMed

    Apell, Hans-Jürgen; Damnjanovic, Bojana

    2016-01-01

    Reconstitution of P-type ATPases in unilamellar liposomes is a useful technique to study functional properties of these active ion transporters. Experiments with such liposomes provide an easy access to substrate-binding affinities of the ion pumps as well as to the lipid and temperature dependence of the pump current. Here, we describe two reconstitution methods by dialysis and the use of potential-sensitive fluorescence dyes to study transport properties of two P-type ATPases, the Na,K-ATPase from rabbit kidney and the K(+)-transporting KdpFABC complex from E. coli. Several techniques are introduced how the measured fluorescence signals may be analyzed to gain information on properties of the ion pumps. PMID:26695029

  8. Arresting a Torsin ATPase Reshapes the Endoplasmic Reticulum*

    PubMed Central

    Rose, April E.; Zhao, Chenguang; Turner, Elizabeth M.; Steyer, Anna M.; Schlieker, Christian

    2014-01-01

    Torsins are membrane-tethered AAA+ ATPases residing in the nuclear envelope (NE) and endoplasmic reticulum (ER). Here, we show that the induction of a conditional, dominant-negative TorsinB variant provokes a profound reorganization of the endomembrane system into foci containing double membrane structures that are derived from the ER. These double-membrane sinusoidal structures are formed by compressing the ER lumen to a constant width of 15 nm, and are highly enriched in the ATPase activator LULL1. Further, we define an important role for a highly conserved aromatic motif at the C terminus of Torsins. Mutations in this motif perturb LULL1 binding, reduce ATPase activity, and profoundly limit the induction of sinusoidal structures. PMID:24275647

  9. Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump.

    PubMed

    Møller, Jesper V; Nissen, Poul; Sørensen, Thomas L-M; le Maire, Marc

    2005-08-01

    The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains. PMID:16009548

  10. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    PubMed

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. PMID:26991466

  11. Copper transporting P-type ATPases and human disease.

    PubMed

    Cox, Diane W; Moore, Steven D P

    2002-10-01

    Copper transporting P-type ATPases, designated ATP7A and ATP7B, play an essential role in mammalian copper balance. Impaired intestinal transport of copper, resulting from mutations in the ATP7A gene, lead to Menkes disease in humans. Defects in a similar gene, the copper transporting ATPase ATP7B, result in Wilson disease. This ATP7B transporter has two functions: transport of copper into the plasma protein ceruloplasmin, and elimination of copper through the bile. Variants of ATP7B can be functionally assayed to identify defects in each of these functions. Tissue expression studies of the copper ATPases and their copper chaperone ATOX1 indicate that there is not complete overlap in expression. Other chaperones may be important for the transport of copper into ATP7A and ATP7B. PMID:12539960

  12. The expression of myosin genes in developing skeletal muscle in the mouse embryo

    SciTech Connect

    Lyons, G.E.; Ontell, M.; Cox, R.; Sassoon, D.; Buckingham, M. )

    1990-10-01

    Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and acculumation.

  13. Effects of Myosin “Essential” Light Chain A1 on the Aggregation Properties of the Myosin Head

    PubMed Central

    Markov, D.I.; Nikolaeva, O.P.

    2010-01-01

    We compared the thermal aggregation properties of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. Temperature dependencies for the aggregation of these two S1 isoforms, as measured by the increase in turbidity, were compared with the temperature dependencies of their thermal denaturation obtained from differential scanning calorimetry (DSC) experiments. At relatively high ionic strength (in the presence of 100 mM KCl) close to its physiological values in muscle fibers, we have found no appreciable difference between the two S1 isoforms in their thermally induced aggregation. Under these conditions, the aggregation of both S1 isoforms was independent of the protein concentration and resulted from their irreversible denaturation, which led to the cohesion of denatured S1 molecules. In contrast, a significant difference between these S1 isoforms was revealed in their aggregation measured at low ionic strength. Under these conditions, the aggregation of S1 containing a light chain A1 (but not A2) was strongly dependent on protein concentration, the increase of which (from 0.125 to 2.0 mg/ml) shifted the aggregation curve by ~10 degrees towards the lower temperatures. It has been concluded that the aggregation properties of this S1 isoform at low ionic strength is basically determined by intermolecular interactions of the N–terminal extension of the A1 light chain (which is absent in the A2 light chain) with other S1 molecules. These interactions seem to be independent of the S1 thermal denaturation, and they may take place even at low temperature. PMID:22649644

  14. Infrared spectroscopic studies on the V-ATPase.

    PubMed

    Kandori, Hideki; Furutani, Yuji; Murata, Takeshi

    2015-01-01

    V-ATPase is an ATP-driven rotary motor that vectorially transports ions. Together with F-ATPase, a homologous protein, several models on the ion transport have been proposed, but their molecular mechanisms are yet unknown. V-ATPase from Enterococcus hirae forms a large supramolecular protein complex (total molecular weight: ~700,000) and physiologically transports Na⁺ and Li⁺ across a hydrophobic lipid bilayer. Stabilization of these cations in the binding site has been discussed on the basis of X-ray crystal structures of a membrane-embedded domain, the K-ring (Na⁺ and Li⁺ bound forms). Sodium or lithium ion binding-induced difference FTIR spectra of the intact E. hirae V-ATPase have been measured in aqueous solution at physiological temperature. The results suggest that sodium or lithium ion binding induces the deprotonation of Glu139, a hydrogen-bonding change in the tyrosine residue and rigid α-helical structures. Identical difference FTIR spectra between the entire V-ATPase complex and K-ring strongly suggest that protein interaction with the I subunit does not cause large structural changes in the K-ring. This result supports the previously proposed Na⁺ transport mechanism by V-ATPase stating that a flip-flop movement of a carboxylate group of Glu139 without large conformational changes in the K-ring accelerates the replacement of a Na⁺ ion in the binding site. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems. PMID:25111748

  15. Flexibility of myosin attachment to surfaces influences F-actin motion.

    PubMed Central

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

    1995-01-01

    We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) 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 (LC2) 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. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement. Images FIGURE 1 FIGURE 4 FIGURE 8 PMID:7544167

  16. Whole genome duplication events in plant evolution reconstructed and predicted using myosin motor proteins

    PubMed Central

    2013-01-01

    Background The evolution of land plants is characterized by whole genome duplications (WGD), which drove species diversification and evolutionary novelties. Detecting these events is especially difficult if they date back to the origin of the plant kingdom. Established methods for reconstructing WGDs include intra- and inter-genome comparisons, KS age distribution analyses, and phylogenetic tree constructions. Results By analysing 67 completely sequenced plant genomes 775 myosins were identified and manually assembled. Phylogenetic trees of the myosin motor domains revealed orthologous and paralogous relationships and were consistent with recent species trees. Based on the myosin inventories and the phylogenetic trees, we have identified duplications of the entire myosin motor protein family at timings consistent with 23 WGDs, that had been reported before. We also predict 6 WGDs based on further protein family duplications. Notably, the myosin data support the two recently reported WGDs in the common ancestor of all extant angiosperms. We predict single WGDs in the Manihot esculenta and Nicotiana benthamiana lineages, two WGDs for Linum usitatissimum and Phoenix dactylifera, and a triplication or two WGDs for Gossypium raimondii. Our data show another myosin duplication in the ancestor of the angiosperms that could be either the result of a single gene duplication or a remnant of a WGD. Conclusions We have shown that the myosin inventories in angiosperms retain evidence of numerous WGDs that happened throughout plant evolution. In contrast to other protein families, many myosins are still present in extant species. They are closely related and have similar domain architectures, and their phylogenetic grouping follows the genome duplications. Because of its broad taxonomic sampling the dataset provides the basis for reliable future identification of further whole genome duplications. PMID:24053117

  17. Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala.

    PubMed

    Lamprecht, R; Margulies, D S; Farb, C R; Hou, M; Johnson, L R; LeDoux, J E

    2006-01-01

    Learning and memory depend on signaling molecules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the training stimuli were presented in a non-associative manner. Anatomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically implicated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nucleus of the amygdala. When ML-7 was applied without associative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the circuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning. PMID:16515842

  18. A role for myosin Va in cerebellar plasticity and motor learning: a possible mechanism underlying neurological disorder in myosin Va disease.

    PubMed

    Miyata, Mariko; Kishimoto, Yasushi; Tanaka, Masahiko; Hashimoto, Kouichi; Hirashima, Naohide; Murata, Yoshiharu; Kano, Masanobu; Takagishi, Yoshiko

    2011-04-20

    Mutations of the myosin Va gene cause the neurological diseases Griscelli syndrome type 1 and Elejalde syndrome in humans and dilute phenotypes in rodents. To understand the pathophysiological mechanisms underlying the neurological disorders in myosin Va diseases, we conducted an integrated analysis at the molecular, cellular, electrophysiological, and behavioral levels using the dilute-neurological (d-n) mouse mutant. These mice manifest an ataxic gait and clonic seizures during postnatal development, but the neurological disorders are ameliorated in adulthood. We found that smooth endoplasmic reticulum (SER) rarely extended into the dendritic spines of Purkinje cells (PCs) of young d-n mice, and there were few, if any, IP(3) receptors. Moreover, long-term depression (LTD) at parallel fiber-PC synapses was abolished, consistent with our previous observations in juvenile lethal dilute mutants. Young d-n mice exhibited severe impairment of cerebellum-dependent motor learning. In contrast, adult d-n mice showed restoration of motor learning and LTD, and these neurological changes were associated with accumulation of SER and IP(3) receptors in some PC spines and the expression of myosin Va proteins in the PCs. RNA interference-mediated repression of myosin Va caused a reduction in the number of IP(3) receptor-positive spines in cultured PCs. These findings indicate that myosin Va function is critical for subsequent processes in localization of SER and IP(3) receptors in PC spines, LTD, and motor learning. Interestingly, d-n mice had defects of motor coordination from young to adult ages, suggesting that the role of myosin Va in PC spines is not sufficient for motor coordination. PMID:21508232

  19. Non-muscle myosin-II-B filament regulation of paracellular resistance in cervical epithelial cells is associated with modulation of the cortical acto-myosin

    PubMed Central

    Li, Xin; Gorodeski, George

    2007-01-01

    Objective To understand myosin regulation of epithelial permeability. Methods Experimental study, using human cervical epithelial cells CaSki. Endpoints were paracellular permeability (determined in terms of transepithelial electrical resistance); non-muscle myosin-II-B (NMM-II-B) cellular localization; NMM-II-B phosphorylation status; NMM-II-B – actin interaction (determined in-vitro by the immunoprecipitation-immunoreactivity method); and NMM-II-B filamentation (determined in-vitro using purified NMM-II-B filaments in terms of filaments disassembly / assembly ratios. Results Treatment of cells with the ROCK inhibitor Y-27632 or with the phosphatase inhibitor okadaic acid decreased the Resistance of the Lateral Intercellular Space (RLIS), and increased phosphorylation of non-muscle myosin-II-B (NMM-II-B) on threonine and serine residues. Y-27632 induced disorganization of the cortical acto-myosin and decreased co-immunoprecipitation of actin with NMM-II-B. Homodimerization assays using NMM-II-B filaments from cells treated with Y-27632 or okadaic acid revealed decreased filamentation compared to control cells. However, okadaic acid blocked Y-27632 decreased filamentation. Treatment with DRB, CK2 inhibitor, induced opposing effects to those of Y-27632 and okadaic acid. Treatment with DRB did not involve modulation of actin depolymerization, suggesting that NMM-II-B regulation of the RLIS was independent of actin polymerization status. Exposure of NMM-II-B filaments to CK2 increased filamentation, regardless of prior treatments in-vivo with Y-27632, okadaic acid, or DRB. Conclusions The results suggest that NMM-II-B filaments are in steady-state equilibrium of phosphorylation-dephosphorylation mediated by CK2 and by ROCK-regulated myosin heavy chain phosphatase, respectively. Increased phosphorylation would tend to inhibit assembly of NMM-II-B filaments and lead to decreased actin-myosin interaction, which would tend to decrease the RLIS and increase the paracellular permeability. PMID:17088080

  20. Heterogeneity of alpha-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins.

    PubMed

    Hoh, Joseph F Y; Withers, Kerry W; Zhong, Wendy W H

    2008-03-01

    The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V(1a), V(1b),V(1c), V(2) and V(3), in order of decreasing mobility. In western blots, V(1a), V(1b), and V(1c) reacted with anti-alpha-MyHC antibody, but not with anti-beta-MyHC, whereas V(2) and V(3) reacted with anti-beta-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-alpha-MyHC antibody while the third bound anti-beta-MyHC antibody. We conclude that V(1a), V(1b), V(1c) are triplets arising from the dimerization of two distinct alpha-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased alpha-MyHC content significantly (t-test, P < 0.001) from 91.6 +/- 5.9% (SEM, n = 4) in control animals to 67.2 +/- 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in beta-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians. PMID:17975714

  1. Na,K-ATPase and epithelial tight junctions.

    PubMed

    Rajasekaran, Sigrid A; Rajasekaran, Ayyappan K

    2009-01-01

    Tight junctions are unique organelles in polarized epithelial and endothelial cells that regulate the flow of solutes and ions across the epithelial barrier. The structure and functions of tight junctions are regulated by a wide variety of signaling and molecular mechanisms. Several recent studies in mammals, drosophila, and zebrafish reported a new role for Na,K-ATPase, a well-studied ion transporter, in the modulation of tight junction development, permeability, and polarity. In this review, we have attempted to compile these new reports and suggest a model for a conserved role of Na,K-ATPase in the regulation of tight junction structure and functions. PMID:19273189

  2. Regulation of cough by neuronal Na(+)-K(+) ATPases.

    PubMed

    Canning, Brendan J; Farmer, David G S

    2015-06-01

    The Na(+)-K(+) ATPases play an essential role in establishing the sodium gradients in excitable cells. Multiple isoforms of the sodium pumps have been identified, with tissue and cell specific expression patterns. Because the vagal afferent nerves regulating cough must be activated at sustained high frequencies of action potential patterning to achieve cough initiation thresholds, it is a certainty that sodium pump function is essential to maintaining cough reflex sensitivities in health and in disease. The mechanisms by which Na(+)-K(+) ATPases regulate bronchopulmonary vagal afferent nerve excitability are reviewed as are potential therapeutic strategies targeting the sodium pumps in cough. PMID:26048736

  3. Vacuolar-type H+-ATPase-mediated proton transport in the rat parietal cell.

    PubMed

    Kopic, Sascha; Wagner, Maximilian E H; Griessenauer, Christoph; Socrates, Thenral; Ritter, Markus; Geibel, John P

    2012-03-01

    The vacuolar-type H-ATPase (V-ATPase) plays an important role in the active acidification of intracellular organelles. In certain specialized cells, such as the renal intercalated cell, apical V-ATPase can also function as a proton secretion pathway. In the parietal cells of the stomach, it has been thought that acid secretion is controlled solely via the H,K-ATPase. However, recent observations suggest that functional V-ATPase is necessary for acid secretion to take place. This study aimed to investigate and characterize the role of V-ATPase in parietal cell proton transport. Individual rat gastric glands were incubated with the pH-sensitive dye (BCECF) to monitor changes in intracellular pH in real time. Parietal cell V-ATPase activity was measured by quantifying the rate of intracellular alkalinization (ΔpH/minute) following an acid load, while excluding the contribution of non-V-ATPase proton transport mechanisms through pharmacological inhibition or ion substitution. Expression of V-ATPase was confirmed by immunohistochemistry. We observed concanamycin A-sensitive V-ATPase activity in rat parietal cells following intracellular acidification and H,K-ATPase inhibition. Furthermore, V-ATPase-mediated proton transport could be abolished by inhibiting trafficking mechanisms with paclitaxel and by stimulating H,K-ATPase with acid secretagogues. Our results propose that parietal cells contain a functional V-ATPase that can be mobilized using a microtubule network. V-ATPase may function as an auxiliary acid secretion or proton-buffering pathway in parietal cells, which is inactive during H,K-ATPase activity. Our findings may have important implications for patients experiencing acid breakthrough under proton pump inhibitor therapy. PMID:22146938

  4. 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.

  5. Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

    PubMed

    Lelli, Andrea; Michel, Vincent; Boutet de Monvel, Jacques; Cortese, Matteo; Bosch-Grau, Montserrat; Aghaie, Asadollah; Perfettini, Isabelle; Dupont, Typhaine; Avan, Paul; El-Amraoui, Aziz; Petit, Christine

    2016-01-18

    The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping. PMID:26754646

  6. Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity

    PubMed Central

    Sen, Arnab; Nagy-Zsvér-Vadas, Zsanett

    2012-01-01

    The assembly and consolidation of the adherens junctions (AJs) are key events in the establishment of an intact epithelium. However, AJs are further modified to obtain flexibility for cell migration and morphogenetic movements. Intact AJs in turn are a prerequisite for the establishment and maintenance of apical–basal polarity in epithelial cells. In this study, we report that the conserved PDZ (PSD95, Discs large, ZO-1) domain–containing protein PATJ (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical–basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation. PATJ directly bound to the Myosin-binding subunit of Myosin phosphatase and decreased Myosin dephosphorylation, resulting in activated Myosin. Thereby, PATJ supports the stability of the Zonula Adherens. Notably, weakening of AJ in a PATJ mutant epithelium led first to a loss of Myosin from the AJ, subsequently to a disassembly of the AJ, and finally, to a loss of apical–basal polarity and disruption of the tissue. PMID:23128243

  7. 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

  8. 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

  9. 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

    2016-01-01

    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

  10. Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

    PubMed Central

    Smith, Tara C.; Fridy, Peter C.; Li, Yinyin; Basil, Shruti; Arjun, Sneha; Friesen, Ryan M.; Leszyk, John; Chait, Brian T.; Rout, Michael P.; Luna, Elizabeth J.

    2013-01-01

    Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis. PMID:24088567

  11. High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

    PubMed Central

    Binder, Benjamin P.; Cornea, Sinziana; Thompson, Andrew R.; Moen, Rebecca J.; Thomas, David D.

    2015-01-01

    Using electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) bound stereospecifically to Dictyostelium myosin II, we determined with high resolution the orientation of individual structural elements in the catalytic domain while myosin is in complex with actin. BSL was attached to a pair of engineered cysteine side chains four residues apart on known α-helical segments, within a construct of the myosin catalytic domain that lacks other reactive cysteines. EPR spectra of BSL-myosin bound to actin in oriented muscle fibers showed sharp three-line spectra, indicating a well-defined orientation relative to the actin filament axis. Spectral analysis indicated that orientation of the spin label can be determined within <2.1° accuracy, and comparison with existing structural data in the absence of nucleotide indicates that helix orientation can also be determined with <4.2° accuracy. We used this approach to examine the crucial ADP release step in myosin’s catalytic cycle and detected reversible rotations of two helices in actin-bound myosin in response to ADP binding and dissociation. One of these rotations has not been observed in myosin-only crystal structures. PMID:26056276

  12. Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin.

    PubMed

    Sato, M; Ye, L H; Kohama, K

    1995-07-01

    Myosin light chain kinase (MLCK) was prepared from smooth muscle of bovine aorta. MLCK inhibited the ATP-dependent movement of actin filaments on a glass surface coated with smooth muscle myosin that had been phosphorylated. The inhibitory effect was abolished by calmodulin in the presence of Ca2+ (Ca-CaM). The abolition was also observed when the concentration of actin filaments was increased. The inhibitory effect and its abolition were related to the actin-binding activity of MLCK, that is antagonized by Ca-CaM. PMID:8537296

  13. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    NASA Astrophysics Data System (ADS)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P < 0.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase Β1, ATPase B2, and ATPase B3 is highly correlated ( P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  14. Cation stoichiometry and cation pathway in the Na,K-ATPase and nongastric H,K-ATPase.

    PubMed

    Horisberger, Jean-Daniel; Guennoun, Saida; Burnay, Muriel; Geering, Kathi

    2003-04-01

    The mechanism of cation translocation by the Na,K-ATPase was investigated by cysteine scanning mutagenesis and measurements of accessibility through exposure to cysteine reagents. In the native protein, accessible residues were found only at the most extracellular residues of the 5th and 6th transmembrane segments (TMS) and the short loop between them. However, after modification by palytoxin a number of residues became accessible along the whole length of the 5th TMS and in the outer half of the 6th TMS, showing the contribution of each of these segments to the "channel" formed by the palytoxin-transformed Na,K-pump. Assuming that this structure is similar in the native and the palytoxin-transformed pump, our data allow us to determine the residues lining the cation pathway from the extracellular solution to their binding sites. A critical position in the 5th TMS contains a lysine conserved in all known nonelectrogenic H,K-ATPases, and a serine in all known electrogenic Na,K-ATPase sequences. Wild-type or mutant Na,K-or H,K-ATPase a subunits were coinjected with the Bufo beta2 subunit in Xenopus oocytes and Rb(86) uptake and electrophysiological measurements were performed. An electrogenic activity was recorded for the H,K-ATPase mutants in which the positively charged lysine had been replaced by neutral or negatively charged residues, while nonelectrogenic transport was observed with the S(782)R mutant of the Na,K-ATPase. The presence or the absence of a positively charged residue at the S(782) position appears to be critical for the stoichiometry of cation exchange. PMID:12763785

  15. Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles.

    PubMed

    Mukherjea, Monalisa; Ali, M Yusuf; Kikuti, Carlos; Safer, Daniel; Yang, Zhaohui; Sirkia, Helena; Ropars, Virginie; Houdusse, Anne; Warshaw, David M; Sweeney, H Lee

    2014-09-11

    It is unclear whether the reverse-direction myosin (myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions. PMID:25159143

  16. A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Steimle, Paul A.; Kent Fulcher, F.; Patel, Yashomati M. . E-mail: ympatel@uncg.edu

    2005-06-17

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis.

  17. Characterization of a NO3−-Sensitive H+-ATPase from Corn Roots 1

    PubMed Central

    O'Neill, Sharman D.; Bennett, Alan B.; Spanswick, Roger M.

    1983-01-01

    When assayed in the presence of azide, NO3− was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 × M017) root microsomal membranes. The distribution of the NO3−-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H+-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 μm vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO3−-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H+ from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K+ > NH4+ > Rb+ > Cs+ > Li+ > Na+ > choline+) whereas the NO3−-sensitive ATPase was stimulated by anions (Cl− > Br− > C2H3O2− > SO42− > I− > HCO3− > SCN−). Both ATPases required divalent cations. However, the order of preference for the NO3−-sensitive ATPase (Mn2+ > Mg2+ > Co2+ > Ca2+ > Zn2+) differed from that of the vanadate-sensitive ATPase (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO3−-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO3−-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO3−-sensitive ATPase was stimulated by gramicidin and was associated with NO3−-inhibitable H+ transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO3−-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO3− sensitivity is a better criterion by which to identify this ATPase than either Cl− stimulation or H+ transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization. PMID:16663096

  18. Loss of myostatin expression alters fiber-type distribution and expression of myosin heavy chain isoforms in slow- and fast-type skeletal muscle.

    PubMed

    Girgenrath, Stefan; Song, Kening; Whittemore, Lisa-Anne

    2005-01-01

    Myostatin (Mstn) is a member of the transforming growth factor-beta family that negatively regulates skeletal muscle mass. Mstn knockout mice have greater skeletal muscle mass than wild-type littermates. We investigated the effect of Mstn on fiber type by comparing adult muscles from the murine Mstn knockout with wild-type controls. Based on myofibrillar ATPase staining, the soleus of Mstn knockout mice displays a larger proportion of fast type II fibers and a reduced proportion of slow type I fibers compared with wild-type animals. Based on staining for succinate dehydrogenase (SDH) activity, a larger proportion of glycolytic fibers and a reduced proportion of oxidative fibers occur in the extensor digitorum longus (EDL) of Mstn knockouts. These differences in distribution of fiber types are accompanied by differences in the expression of myosin heavy chain (MHC) isoforms. In both Mstn knockout soleus and EDL, larger numbers of faster MHC isoforms are expressed at the expense of slower isoforms when compared with wild-type littermates. Thus, the absence of Mstn in the knockout mouse leads to an overall faster and more glycolytic muscle phenotype. This muscle phenotype is likely a consequence of developmental processes, and inhibition of Mstn in adults does not cause a transformation to a more fast and glycolytic phenotype. Our findings suggest that myostatin has a critical role in regulating the formation, proliferation, or differentiation of fetal myoblasts and postnatal fibers. PMID:15468312

  19. Genome-wide identification, splicing, and expression analysis of the myosin gene family in maize (Zea mays)

    PubMed Central

    Wang, Guifeng; Zhong, Mingyu; Wang, Gang; Song, Rentao

    2014-01-01

    The actin-based myosin system is essential for the organization and dynamics of the endomembrane system and transport network in plant cells. Plants harbour two unique myosin groups, class VIII and class XI, and the latter is structurally and functionally analogous to the animal and fungal class V myosin. Little is known about myosins in grass, even though grass includes several agronomically important cereal crops. Here, we identified 14 myosin genes from the genome of maize (Zea mays). The relatively larger sizes of maize myosin genes are due to their much longer introns, which are abundant in transposable elements. Phylogenetic analysis indicated that maize myosin genes could be classified into class VIII and class XI, with three and 11 members, respectively. Apart from subgroup XI-F, the remaining subgroups were duplicated at least in one analysed lineage, and the duplication events occurred more extensively in Arabidopsis than in maize. Only two pairs of maize myosins were generated from segmental duplication. Expression analysis revealed that most maize myosin genes were expressed universally, whereas a few members (XI-1, -6, and -11) showed an anther-specific pattern, and many underwent extensive alternative splicing. We also found a short transcript at the O1 locus, which conceptually encoded a headless myosin that most likely functions at the transcriptional level rather than via a dominant-negative mechanism at the translational level. Together, these data provide significant insights into the evolutionary and functional characterization of maize myosin genes that could transfer to the identification and application of homologous myosins of other grasses. PMID:24363426

  20. Myosin VA Movements in Normal and Dilute-Lethal Axons Provide Support for a Dual Filament Motor Complex

    PubMed Central

    Bridgman, P.C.

    1999-01-01

    To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va–associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein–myosin Va tail construct. Myosin Va–associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va null) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va–associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules. PMID:10477758

  1. Electrophysiological characterization of ATPases in native synaptic vesicles and synaptic plasma membranes.

    PubMed

    Obrdlik, Petr; Diekert, Kerstin; Watzke, Natalie; Keipert, Christine; Pehl, Ulrich; Brosch, Catrin; Boehm, Nicole; Bick, Inga; Ruitenberg, Maarten; Volknandt, Walter; Kelety, Bela

    2010-04-01

    Vesicular V-ATPase (V-type H+-ATPase) and the plasma membrane-bound Na+/K+-ATPase are essential for the cycling of neurotransmitters at the synapse, but direct functional studies on their action in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed SSM (solid supported membrane)-based electrophysiological analyses of synaptic vesicles and plasma membranes prepared from rat brains by sucrose-gradient fractionation. Acidification experiments revealed V-ATPase activity in fractions containing the vesicles but not in the plasma membrane fractions. For the SSM-based electrical measurements, the ATPases were activated by ATP concentration jumps. In vesicles, ATP-induced currents were inhibited by the V-ATPase-specific inhibitor BafA1 (bafilomycin A1) and by DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate). In plasma membranes, the currents were inhibited by the Na+/K+-ATPase inhibitor digitoxigenin. The distribution of the V-ATPase- and Na+/K+-ATPase-specific currents correlated with the distribution of vesicles and plasma membranes in the sucrose gradient. V-ATPase-specific currents depended on ATP with a K0.5 of 51+/-7 microM and were inhibited by ADP in a negatively co-operative manner with an IC50 of 1.2+/-0.6 microM. Activation of V-ATPase had stimulating effects on the chloride conductance in the vesicles. Low micromolar concentrations of DIDS fully inhibited the V-ATPase activity, whereas the chloride conductance was only partially affected. In contrast, NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] inhibited the chloride conductance but not the V-ATPase. The results presented describe electrical characteristics of synaptic V-ATPase and Na+/K+-ATPase in their native surroundings, and demonstrate the feasibility of the method for electrophysiological studies of transport proteins in native intracellular compartments and plasma membranes. PMID:20100168

  2. Molecular Cloning of Tomato Plasma Membrane H+-ATPase 1

    PubMed Central

    Ewing, Nicholas N.; Wimmers, Larry E.; Meyer, David J.; Chetelat, Roger T.; Bennett, Alan B.

    1990-01-01

    Two cDNA clones (LHA1 and LHA2) from tomato (Lycopersicon esculentum) which likely encode isoforms of the plasma membrane H+-ATPase were isolated. The longest cDNA (3229 base pairs), LHA1, comprises an open reading frame that encodes a 956 amino acid, 105 kilodalton polypeptide with several potential transmembrane domains. In vitro transcription and translation of LHA1 yields a major translation product of approximately 100 kilodaltons that is immunoprecipitable with antiserum to the corn root plasma membrane H+-ATPase. LHA2 encodes a portion of a coding sequence that is 96% identical to LHA1, suggesting that LHA2 encodes an isoform of the H+-ATPase. Genomic DNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to a common set of six to eight restriction fragments at moderate stringency and to single distinct fragments at high stringency. LHA1 and LHA2 map to distinct sites on chromosomes three and six, respectively. RNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to 3.4 kilobase pair transcripts present in both leaves and roots, although the LHA2 transcript is relatively more abundant in leaves than in roots. These results indicate that in tomato as many as six to eight genes may encode the plasma membrane H+-ATPase, two of which are expressed at the level of mRNA in both roots and leaves. Images Figure 3 Figure 4 Figure 5 Figure 7 PMID:16667929

  3. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  4. Fluoride inhibition of proton-translocating ATPases of oral bacteria.

    PubMed Central

    Sutton, S V; Bender, G R; Marquis, R E

    1987-01-01

    The ATPases of isolated membranes of lactic acid bacteria were found to be inhibited by fluoride in a complex manner. Among the enzymes tested, that of Streptococcus mutans GS-5 was the most sensitive to fluoride, and the initial rate of hydrolysis of ATP was reduced 50% by approximately 3 mM fluoride. The enzyme of Lactobacillus casei ATCC 4646 was the most resistant, and about 25 mM fluoride was required for 50% inhibition. The response to fluoride appeared to involve reversible, noncompetitive inhibition during short exposure to low levels of fluoride and nonreversible inhibition at higher fluoride levels. In addition, kinetic studies of the effects of fluoride on the enzymes of membranes of S. mutans and L. casei indicated that reversible inhibition was at least partly overcome at high levels of either ATP or Mg. The effects of pH on fluoride inhibition of ATPases were markedly different from the effects of pH on inhibition of acid/base regulation of intact cells by fluoride. It appeared that formation of HF was not required for inhibition of the ATPases. F1 ATPases isolated from the membranes by washing with buffers of low ionic strength proved to be less sensitive to fluoride than the membrane-associated F1F0 holoenzymes, and it was concluded that the F0 or membrane sector of the holoenzyme is involved in fluoride inhibition. PMID:2889674

  5. Crystal Structure of the Vanadate-Inhibited Ca(2+)-ATPase.

    PubMed

    Clausen, Johannes D; Bublitz, Maike; Arnou, Bertrand; Olesen, Claus; Andersen, Jens Peter; Møller, Jesper Vuust; Nissen, Poul

    2016-04-01

    Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca(2+)-ATPase with bound vanadate in the absence of Ca(2+). Vanadate is bound at the catalytic site as a planar VO3(-) in complex with water and Mg(2+) in a dephosphorylation transition-state-like conformation. Validating bound VO3(-) by anomalous difference Fourier maps using long-wavelength data we also identify a hitherto undescribed Cl(-) site near the dephosphorylation site. Crystallization was facilitated by trinitrophenyl (TNP)-derivatized nucleotides that bind with the TNP moiety occupying the binding pocket that normally accommodates the adenine of ATP, rationalizing their remarkably high affinity for E2P-like conformations of the Ca(2+)-ATPase. A comparison of the configurations of bound nucleotide analogs in the E2·VO3(-) structure with that in E2·BeF3(-) (E2P ground state analog) reveals multiple binding modes to the Ca(2+)-ATPase. PMID:27050689

  6. Calcium-ATPases: Gene disorders and dysregulation in cancer.

    PubMed

    Dang, Donna; Rao, Rajini

    2016-06-01

    Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26608610

  7. A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton.

    PubMed

    Yuan, Yanggang; Zhao, Chuanyan; An, Xiaofei; Wu, Lin; Wang, Hui; Zhao, Min; Bai, Mi; Duan, Suyan; Zhang, Bo; Zhang, Aihua; Xing, Changying

    2016-06-01

    Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. PMID:26902808

  8. 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. © 2015 Wiley Periodicals, Inc. PMID:26492945

  9. 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.

  10. Increased activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the frontal cortex and cerebellum of autistic individuals

    PubMed Central

    Ji, Lina; Chauhan, Abha; Brown, W. Ted; Chauhan, Ved

    2009-01-01

    Aims Na+/K+-ATPase and Ca2+/Mg2+-ATPase are enzymes known to maintain intracellular gradients of ions that are essential for signal transduction. The aim of this study was to compare the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in post-mortem brain samples from the cerebellum and frontal, temporal, parietal, and occipital cortices from autistic and age-matched control subjects. Main methods The frozen postmortem tissues from different brain regions of autistic and control subjects were homogenized. The activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase were assessed in the brain homogenates by measuring inorganic phosphorus released by the action of Na+/K+- and Ca2+/Mg2+- dependent hydrolysis of ATP. Key findings In the cerebellum, the activities of both Na+/K+-ATPase and Ca2+/Mg2+-ATPase were significantly increased in the autistic samples compared with their age-matched controls. The activity of Na+/K+-ATPase but not Ca2+/Mg2+-ATPase was also significantly increased in the frontal cortex of the autistic samples as compared to the age-matched controls. In contrast, in other regions, i.e., the temporal, parietal and occipital cortices, the activities of these enzymes were similar in autism and control groups. Significance The results of this study suggest brain-region specific increases in the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in autism. Increased activity of these enzymes in the frontal cortex and cerebellum may be due to compensatory responses to increased intracellular calcium concentration in autism. We suggest that altered activities of these enzymes may contribute to abnormal neuronal circuit functioning in autism. PMID:19863947

  11. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

    PubMed

    Šubrtová, Karolína; Panicucci, Brian; Zíková, Alena

    2015-02-01

    In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells. PMID:25714685

  12. ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component, Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

    PubMed Central

    Šubrtová, Karolína; Panicucci, Brian; Zíková, Alena

    2015-01-01

    In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψm) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells. PMID:25714685

  13. Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

    NASA Technical Reports Server (NTRS)

    Kristjansson, H.; Hochstein, L. I.

    1986-01-01

    Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

  14. Decreased ATPase activity in adriamycin nephrosis is independent of proteinuria

    SciTech Connect

    Bakker, W.W.; Kalicharan, D.; Donga, J.; Hulstaert, C.E.; Hardonk, M.J.

    1987-03-01

    In previous studies from this laboratory it has been shown that ATP-ase activity in situ in the glomerular basement membrane (GBM) is clearly reduced in rats rendered nephrotic after treatment with adriamycin (ADR). The question was raised whether this reduction of ATP-ase activity in the GBM is due to toxic activity of ADR or rather a result of the nephrotic condition per se. Therefore, we studied ATP-ase activity using the cerium-based method in kidneys from ADR-treated rats without proteinuria (48 hr after ADR injection), or with proteinuria (approximately 150 mg/24 hr) several weeks after ADR injection. Also kidneys from rats rendered nephrotic by surgical ablation and from non-nephrotic rats treated with local X-irradiation (2000 rads) as well as from normal control rats were studied. The results show that in the GBM of ADR-treated or irradiated rats, clear reduction of ATP-ase activity is observed irrespective of their proteinuria, whereas in the GBM of rats rendered nephrotic by renal ablation (approximately 156 mg/24 hr mean protein excretion) no reduction of enzyme activity is found. It is concluded that decreased ATP-ase activity of the glomerular filtration barrier in ADR-treated rats is due to an early toxic activity of this dru